Life Cycle and Biology of Malaria Parasites

2.1 Exo-erythrocytic cycle

An infected female Anopheles mosquito carries malaria parasites – Plasmodium spp. during a blood meal by inoculating sporozoites, the infective stage for humans.

Sporozoites are introduced while the mosquito probes for blood vessels and injects saliva into the skin. These slender, motile forms of the parasite measure approximately 10–15 µm in length and initiate human infection.

Typically, a relatively small number of sporozoites (approximately 8–15) are inoculated, although higher numbers may occasionally be transmitted. Within approximately 45 minutes, they traverse the vascular endothelial layer, cross the sinusoidal cell barrier, and invade hepatocytes, thereby initiating the exoerythrocytic (hepatic) stage of the Plasmodium life cycle. Sporozoites that fail to invade hepatocytes are rapidly cleared from the circulation and do not survive [11–16]. The exact route Plasmodium sporozoites take to hepatocytes has been extensively studied in cell biology. Although hepatocytes lie beneath an endothelial cell lining, the liver is unique in that its endothelial cells have open fenestrations, allowing direct contact between the circulatory system and hepatocytes. However, estimates indicate that the diameter of these fenestrations is 0.1 µm, about ten times smaller than the diameter of a sporozoite [17]. Biochemical and physiological studies have suggested that sporozoites enter the liver by passing through Kupffer cells, which are thought to serve as a gateway for sporozoite entry into the liver [14, 18]. Inside hepatocytes, the parasites undergo asexual replication known as exoerythrocytic schizogony (the hepatic stage). The length of the exoerythrocytic phase and the merozoite output of each infected hepatocyte are species-specific characteristics.

P. vivax completes hepatic development in approximately 6–8 days, producing about 10,000 merozoites per infected hepatocyte. P. ovale develops in about 9 days, yielding approximately 15,000 merozoites. P. malariae requires 12–16 days and produces roughly 2,000 merozoites. P. falciparum matures within 5–7 days and may generate up to 40,000 merozoites per hepatocyte. Upon rupture of the infected hepatocyte, mature hepatic schizonts release merozoites into the bloodstream, thereby initiating the erythrocytic stage of infection [19, 20]. The interval between infection and the onset of clinical manifestations is largely determined by the duration of hepatic development and the subsequent expansion of parasites during the erythrocytic cycle. In Plasmodium vivax and P. ovale infections, a proportion of intrahepatic parasites do not immediately undergo development but instead persist as dormant forms known as hypnozoites. These latent stages can reactivate weeks to months later, initiating renewed hepatic schizogony and leading to a clinical relapse [20]. The ability of P. vivax to establish dormant hypnozoites in hepatocytes enables the parasite to cause recurrent clinical episodes long after the initial infection has resolved. Because of its capacity to produce relapses, P. vivax presents greater obstacles to disease control and elimination than P. falciparum.

In malaria-endemic settings, relapses associated with P. vivax contribute substantially to disease burden in young children and play a key role in sustaining transmission. Similar relapse patterns occur in infections due to P. ovale and various simian malaria species, most notably P. cynomolgi, a species extensively used as an animal model for investigating vivax malaria [21].

Hypnozoite stages are absent in P. falciparum, P. malariae, and P. knowlesi. Hypnozoites have not been demonstrated; therefore, these species are not capable of causing relapse [16, 22]. However, untreated P. falciparum infection may recur within 1–2 years due to persistent low-level parasitemia associated with continued erythrocytic replication, which may remain clinically silent for prolonged periods. In the case of P. malariae, recrudescence has been documented decades after the initial infection, with recurrences reported up to 30 years later or longer [23]. In all the above-mentioned infections, treatment aimed at eliminating circulating erythrocytic stages is sufficient, as no dormant hepatic forms are present.

Earlier hypotheses suggested that malaria relapse resulted from persistent exoerythrocytic development within the liver. These latent parasites persist in hepatocytes without undergoing immediate replication and may reactivate weeks to months (or even years) after the primary infection, initiating a new cycle of hepatic schizogony and subsequent erythrocytic infection [24]. For many years, the biological basis of malaria relapse remained unclear, and recurrent episodes were believed to result from persistent exoerythrocytic development within the liver. This concept was fundamentally revised following the discovery of hypnozoites, dormant liver-stage forms that are unique to P. vivax and P. ovale. After sporozoites invade hepatocytes, only a proportion differentiates into hypnozoites, whereas the remainder undergo normal hepatocytic development, forming replicating schizonts. Hypnozoites persist in a metabolically dormant state within hepatocytes and may reactivate after variable periods of dormancy, initiating hepatic schizogony and the subsequent release of merozoites into the bloodstream, thereby triggering a new erythrocytic cycle. Although considerable progress has been made in understanding hypnozoite biology, the molecular and cellular mechanisms regulating their formation, maintenance, and reactivation remain largely unresolved, primarily because of the limited availability of physiologically relevant experimental models. Consequently, conventional blood-stage antimalarial drugs effectively eliminate circulating parasites and resolve the acute clinical infection but fail to eradicate dormant hepatic forms. Therefore, radical cure of P. vivax and P. ovale malaria requires treatment with hypnozoitocidal agents to eliminate the latent liver reservoir and prevent subsequent relapses [25].

2.2 Erythrocytic cycle

Once motile merozoites, which are morphologically distinct from sporozoites but similarly adapted for rapid invasion, are released from hepatic schizonts, they rapidly invade red blood cells and initiate the erythrocytic stage. Invasion of erythrocytes involves a complex sequence of events that begins with contact between the parasite and specific receptors on the red blood cell membrane. The invasion process involves a highly coordinated sequence of events that begins with attachment of the merozoite to the erythrocyte surface and proper orientation of its apical complex toward the host cell membrane. The parasite actively penetrates the erythrocyte by first reorienting itself, then forming a tight junction, followed by gradual uptake as the erythrocyte membrane invaginates around it. Invasion culminates in the establishment of a parasitophorous vacuole that houses the intracellular parasite [24]. Within the erythrocyte cytosol, the parasite is bounded internally by its plasma membrane and externally by the parasitophorous vacuolar membrane formed during entry. It has been proposed that nutrient exchange between the parasite and the host cytoplasm occurs through specialized channels or pores within the vacuolar membrane. The attachment of the merozoite to erythrocytes is mediated by specific parasite ligands, many of which belong to erythrocyte-binding protein families localized within the micronemes of the apical complex.

These parasites' ligands bind to specific receptors expressed on the surface of erythrocytes [26, 27].

In P. vivax, invasion depends on interaction with the Duffy blood group antigen (Fy^a or Fy^b) on the erythrocyte surface. The high prevalence of the Duffy-negative phenotype among populations in West Africa explains the marked resistance to P. vivax infection and the historically low prevalence of vivax malaria in this region. In P. falciparum, considerable attention has focused on the merozoite ligand EBA-175, a member of the Duffy-binding-like (DBL) superfamily of proteins that mediates binding to host erythrocyte receptors [28–30]. The specific erythrocyte receptors involved in invasion by P. malariae and P. ovale remain incompletely characterized. Studies have demonstrated that Duffy negativity is not an absolute barrier to P. vivax infection. Although the classical model proposed that P. vivax merozoites require the Duffy antigen receptor for chemokines (DARC/ACKR1) on the erythrocyte surface to invade red blood cells, numerous molecular and epidemiological studies have documented P. vivax infections in Duffy-negative individuals, particularly in sub-Saharan Africa. These findings indicate that Duffy negativity confers substantial but incomplete protection and suggest the existence of alternative invasion pathways. Such observations challenge the long-standing paradigm of complete Duffy-mediated resistance and indicate that the epidemiology of vivax malaria may be broader than previously assumed [31].

Following parasite entry, the erythrocyte undergoes rapid and profound structural and biochemical changes. The parasite enters the cell through endocytic invagination of the erythrocyte membrane. Approximately 12–14 hours after invasion by P. falciparum, a species-specific high-molecular-weight antigen, PfEMP1, is expressed on the erythrocyte surface, where it mediates adhesion to the vascular endothelium. PfEMP1 expression reaches its peak in the middle of the erythrocytic cycle (around 24 hours). These infected erythrocytes sequester in the microvasculature and adhere to the endothelium of venules and capillaries in vital organs. This process is referred to as sequestration. The other major human malaria species (P. vivax, P. ovale, and P. malariae) do not exhibit such marked cytoadherence, and all stages of parasite development occur in the peripheral blood [16].

Inside the erythrocyte, the parasite begins to grow, initially forming the ring-like early trophozoites and eventually enlarging to fill the cell. The parasite then undergoes asexual division and becomes a schizont composed of merozoites. During the early intraerythrocytic developmental phase (within approximately the first 12 hours after invasion), small ring forms of the four major human malaria species can be identified microscopically. These early trophozoite stages appear as delicate ring-shaped structures; in Plasmodium falciparum, they often resemble a pair of “headphones,” characterized by a central vacuole surrounded by a thin rim of cytoplasm and one or two dense chromatin dots. The parasites exhibit active cytoplasmic movement within the erythrocyte.

During its maturation, the parasite progressively increases in size and utilizes hemoglobin from the host erythrocyte as a nutrient source. The breakdown of hemoglobin leads to the production of hemozoin, the characteristic pigment associated with malaria infection.

However, the liberated heme moiety presents a toxic challenge. Once released from globin, free heme is oxidized to ferric (Fe³⁺) heme, which is highly reactive and cytotoxic. To detoxify free heme, the parasite converts it into hemozoin, an insoluble microcrystalline pigment. This process involves biomineralization and crystallization of heme into an insoluble form, thereby preventing oxidative damage [31]. Importantly, inhibition of heme detoxification is a major target of several antimalarial drugs. For example, chloroquine and other quinoline-based compounds act in the parasite’s food vacuole by blocking the conversion of heme into hemozoin. This results in the buildup of toxic free heme, which ultimately leads to parasite death. Resistance to chloroquine in P. falciparum is associated with altered drug transport within the food vacuole, which reduces intracellular drug accumulation [31]. As P. vivax grows, the infected erythrocyte enlarges, and red granules known as Schüffner’s dots become visible in the cytoplasm. Schüffner’s dots are also prominent in P. ovale, which likewise alters the shape of the erythrocyte. P. malariae produces a characteristic band form and is usually associated with low parasitemia.

Markedly higher parasitemia (usually above 2%) is most often associated with P. falciparum. About 36 hours following merozoite entry (and roughly 54 hours in P. malariae), multiple rounds of nuclear replication lead to the development of a segmented schizont stage, also referred to, more precisely, as a meront.

The growing parasite eventually occupies the entire interior of the erythrocyte, which becomes spherical, rigid, depleted of hemoglobin, and filled with merozoites. The erythrocyte subsequently ruptures, releasing 6–36 merozoites and destroying the remainder of the red blood cell. The released merozoites rapidly invade other erythrocytes and initiate another asexual cycle. The infection therefore progresses logarithmically, increasing approximately tenfold with each cycle. The asexual erythrocytic cycle lasts 48 hours in P. falciparum, P. vivax, and P. ovale; 72 hours in P. malariae; and 24 hours in P. knowlesi [32].

The erythrocytic cycle is completed when the infected red blood cell ruptures, releasing merozoites that invade additional erythrocytes [16, 24].

Infection with merozoites may also occur through transfusion of blood from an infected donor or via shared contaminated needles, especially among people who inject drugs. Malaria acquired through these routes is referred to as induced or transfusion-related malaria [26].

Congenital malaria is defined as the presence of malarial parasites demonstrated in a peripheral blood smear of a newborn between 24 hours and seven days of life [33]. In modern transfusion medicine, minimizing the risk of transfusion-transmitted malaria (TTM) is an important component of blood safety. Because Plasmodium parasites can survive in donated blood and be transmitted to recipients, many countries implement rigorous donor screening and deferral policies. For example, current guidelines recommend deferring donors with a recent history of malaria, or recent visiting of malaria-endemic areas, also residence in endemic regions for certain time before donation. In some settings, at-risk donations are selectively tested using sensitive assays, such as nucleic acid tests (NATs), to detect Plasmodium infection [34, 35]. Such measures have made TTM extremely rare in countries with comprehensive screening programs, with reported rates of fewer than one case per million transfused blood units in the United States.

4.1 Plasmodium falciparum Welch, 1898 (malignant tertian malaria)

P. falciparum is a unicellular eukaryotic protozoan parasite responsible for causing the most virulent and potentially fatal form of human malaria. It is the predominant species associated with severe clinical manifestations, including cerebral malaria, severe anemia, and multi-organ complications.

Historically referred to as “aestivo-autumnal” or malignant tertian malaria, the infection caused by P. falciparum is now commonly termed P. malaria. The parasite’s ability to undergo extensive asexual replication within human red blood cells underlies its high pathogenicity, making it the leading cause of severe malaria and the predominant driver of global malaria-associated morbidity and mortality.

Malaria causes hundreds of millions of infections and more than half a million deaths annually, with approximately 94–95% of cases and deaths occurring in sub-Saharan Africa, where P. falciparum predominates and remains the leading cause of malaria-related mortality [1, 16, 19].

One of the distinctive biological attributes of P. falciparum is its ability to infect erythrocytes of all ages. This unrestricted invasion capacity promotes extensive parasite replication and contributes to the elevated parasite densities commonly observed in severe and life-threatening malaria. Infected erythrocytes generally retain their normal size, and multiple parasites within a single erythrocyte are commonly observed on peripheral blood smears. Early trophozoite stages appear as delicate ring forms, often containing two chromatin dots, a characteristic morphological feature of the species (Figure 2).

During erythrocytic development, P. falciparum triggers structural and biochemical modifications of the erythrocyte membrane that enhance the binding of infected erythrocytes to endothelial cells lining the microvasculature. This process, termed cytoadherence, contributes significantly to microvascular obstruction and disease severity. This adhesive interaction results in the sequestration of mature P. falciparum-infected erythrocytes within capillaries and postcapillary venules of various organs, particularly the brain, heart, liver, and, in pregnancy including placenta. As a consequence, peripheral blood films typically contain predominantly ring-stage trophozoites and mature gametocytes, whereas later asexual developmental stages, including trophozoites and schizonts, are generally absent from the peripheral circulation. The detection of circulating schizonts is relatively rare and is often associated with severe disease, high parasite densities, and compromised sequestration efficiency [16, 19, 36]. When present, schizonts usually contain 8–32 merozoites, with an average of approximately 20 merozoites per schizont. In P. falciparum infection, the lysis of infected erythrocytes and liberation of merozoites are not fully synchronized across the parasite population. Consequently, patients experience recurrent fever paroxysms, generally occurring at intervals of 48–72 hours. The gametocytes of P. falciparum are morphologically distinctive and exhibit the characteristic crescent or banana-shaped appearance typical for the species while remaining enclosed within the erythrocyte membrane.

In general, macrogametocytes exceed microgametocytes in size, measuring approximately 12–14 µm and 9–11 µm in length, respectively. During gametocyte maturation, hemozoin pigment accumulates near the nucleus, accompanied by characteristic parasite-induced erythrocyte membrane structures, referred to as Maurer’s dots. Gametocytogenesis occurs within infected erythrocytes and requires approximately 10 days for completion. Following commitment to sexual differentiation, mature gametocytes become infectious to Anopheles mosquitoes after approximately 10–12 days. Once mature, they may persist in the peripheral circulation for several days, thereby facilitating transmission to feeding mosquito vectors [16, 19, 36]. In contrast to P. vivax and P. ovale, P. falciparum does not produce dormant hepatic stages (hypnozoites) and therefore does not cause true relapse. Consequently, once blood-stage parasites are eliminated through effective antimalarial treatment or host immune clearance, renewed infection from latent liver stages does not occur. Despite the absence of hypnozoite-mediated relapse, P. falciparum malaria can recur through recrudescence when treatment fails to completely clear erythrocytic parasites. Recrudescence is characterized by the re-emergence of detectable parasitemia arising from persistent low-level blood-stage infection. Recrudescence may occur weeks to months after the initial clinical episode and, in rare cases, has been documented up to approximately two years after the primary infection [16, 19].

4.2 Plasmodium vivax Grassi and Filett, 1889

P. vivax is the leading cause of malaria in regions beyond sub-Saharan Africa and is responsible for a substantial proportion of global malaria cases. In comparison with P. falciparum, it generally produces milder clinical manifestations and lower mortality rates. This perceived lower pathogenicity has contributed to reduced research emphasis and comparatively limited investment in targeted drug and vaccine development. The disease caused by this parasite is often referred to as vivax malaria, or historically, benign tertian malaria.

In a blood smear from a patient infected with P. vivax, various asexual stages and gametocytes may be present. The stages of the asexual cycle depend on when the blood was drawn in relation to the febrile cycle. The febrile paroxysm follows the somewhat synchronous rupture of most of the infected cells, and the released merozoites invade new erythrocytes.

Within several hours after a febrile paroxysm, the majority of infected erythrocytes contain very early developmental forms of the parasite, known as ring-stage trophozoites. In a properly prepared and Giemsa-stained thin blood smear, these parasites appear as delicate blue cytoplasmic rings with a small red chromatin dot, situated within the pale cytoplasm of the erythrocyte [16, 40–42].

Occasionally, parasites may be observed at the periphery of the red blood cell, closely opposed to the cell membrane; this morphological appearance is referred to as an appliqué (accolé – from the French, accolé, meaning “applied” or “attached”). Shortly thereafter, a prominent central vacuole develops within the parasite cytoplasm, displacing the chromatin toward the periphery. At this stage, the parasite resembles a signet ring and has increased in size, often occupying approximately one-third of the infected erythrocyte. During the growth phase, the trophozoite exhibits marked amoeboid motility, assuming irregular and sometimes bizarre shapes. This characteristic amoeboid activity gave rise to the species name vivax, derived from the Latin word meaning “lively” or “energetic” [40–42].

Between 6 and 24 hours after the beginning of the erythrocytic cycle, the trophozoites grow to half the size of the infected cell, and brownish pigment granules appear within them. At the same time, the infected cell appears enlarged, pale, and may contain very fine reddish granules, known as Schüffner’s dots (Figure 3). The exact origin of these dots remains incompletely understood, but, if present, it is evidence of infection with P. vivax or P. ovale. The parasite continues to enlarge over the next 24 hours, eventually occupying nearly the entire erythrocyte, which may be enlarged to 10 to 12 µm [39, 41].

After about 40 hours, the adult trophozoite reduces its amoeboid activity and becomes compact, sometimes appearing smaller than the active, motile stage that preceded it. A single nucleus divides repeatedly, producing 12 to 24 nuclear masses. During the division stage, the parasite is called a schizont. The parasite cytoplasm gradually segments around each nucleus, forming daughter cells known as merozoites. After approximately 44–48 hours, rupture of the infected erythrocyte releases the merozoites, which can then invade other erythrocytes [16, 19, 41].

The erythrocytic developmental cycle is often asynchronous, resulting in the concurrent presence of multiple parasite stages within a single peripheral blood sample. In addition to asexual parasites, gametocytes may be present and are often more readily observed after a malarial febrile episode. Gametocytes mature more slowly than asexual stages and do not exhibit amoeboid activity. They accumulate greater amounts of malarial pigment (hemozoin) within their cytoplasm. Parasitized red blood cells frequently become enlarged and can develop characteristic Schüffner’s dots (red granules) on the surface. They become irregular in shape and lose their red color. Mature gametocytes are large enough to occupy nearly the entire infected erythrocyte. Microgametocytes (male) and macrogametocytes (female) can be distinguished morphologically. The nucleus of the macrogametocyte is small, dense, and compact, whereas the nucleus of the microgametocyte is larger, more diffuse, and paler in appearance [16, 41–43].

All parasite stages visible in thin blood smears can also be observed in thick blood films, although the morphology of the parasites may appear somewhat distorted due to hemolysis of erythrocytes during preparation. Early trophozoites are typically ring-shaped, but in thick smears they often appear partially damaged or irregular. The chromatin may be seen as small red dots associated with clumps of blue cytoplasm. These altered early trophozoites are sometimes referred to as “comet” or “bird-like” forms. Based solely on altered early ring forms in thick films, reliable distinction between P. ovale and P. falciparum may be difficult.

Thick blood films are characterized by the destruction of erythrocytes, resulting in parasite forms being visualized against a background of erythrocyte remnants. In cases of P. vivax and P. ovale malaria, residual cytoplasmic material from parasitized erythrocytes may retain visible Schüffner’s dots, providing an additional morphological clue for species identification.

Gametocytes of P. vivax, P. ovale and P. malariae are morphologically similar, although those of P. malariae tend to be slightly smaller, darker, and lack Schüffner’s dots. For accurate species identification, the evaluation of asexual stages in well-prepared thin blood films is generally more reliable [16, 41–43].

P. vivax can cause malaria relapse through reactivation of dormant hepatic stages (hypnozoites) after a variable latent period. Relapses may occur from weeks to months, and in some cases up to five years, after the initial infection. The frequency and timing of relapses vary among geographically distinct parasite strains, with some strains causing frequent early relapses, whereas others are characterized by prolonged latency intervals.

In addition to relapse, P. vivax infection may also exhibit recrudescence, which results from the persistence of low-level blood-stage parasitemia due to erythrocytic forms that were not eliminated [16, 41–43].

4.3 Plasmodium ovale – Stephens, 1922

P. ovale has long been recognized as a human malaria parasite, although its taxonomic classification has been revised in recent decades. It is mostly present in tropical Africa, mainly in West Africa, where the high frequency of the Duffy-negative blood group phenotype markedly reduces transmission of P. vivax. In these regions, P. ovale partially occupies the ecological niche typical for P. vivax. Besides Africa, P. ovale has been reported sporadically in Southeast Asia, the Western Pacific region, and occasionally in South America.

Recently, P. ovale has been divided into two molecularly distinguished but morphologically similar species, i.e., P. ovale curtisi and P. ovale wallikeri. These species differ genetically but share similar clinical and epidemiological characteristics. P. ovale infection produces a tertian fever pattern (48-hour periodicity), clinically resembling P. vivax malaria, although it generally follows a milder course. Relapses are well documented and occur through mechanisms similar to those observed in P. vivax, as P. ovale also forms hepatic hypnozoites. The morphological features that originally led to recognition of P. ovale as a distinct species include the characteristic oval or slightly elongated shape of infected erythrocytes, often with fimbriated (ragged) edges. However, this feature is variable and may not be evident in thin blood smears that dry slowly under conditions of high humidity. Additional morphological features may assist in distinguishing P. ovale from P. vivax. The trophozoites of P. ovale are characterized by a more compact shape and show less amoeboid activity than those of P. vivax. Furthermore, the nuclei of the developmental stages tend to appear larger and more compact than those of P. vivax. Hemozoin accumulation is typically less pronounced than in P. vivax. During schizogony, P. ovale usually produces 4–12 merozoites, averaging approximately 8 per schizont, similar to P. malariae. Rarely, higher numbers (up to 12–16 or slightly more) may be observed [16, 41–43]. In P. ovale infection, infected erythrocytes are typically enlarged and pale. When properly stained, Schüffner’s dots are often more prominent and more readily visible than in P. vivax infection. The margins of infected erythrocytes are frequently irregular, appearing fimbriated, elongated, oval, or otherwise distorted in shape. Parasites in thick blood films resemble those seen in P. vivax infection. However, if schizonts larger than those of P. malariae are identified but contain no more than approximately 12 merozoites and are associated with recognizable Schüffner’s stippling, a presumptive diagnosis of P. ovale may be made [16, 37, 41, 42].

4.4 Plasmodium malariae – Laveran, 1881

The species was recognized as a separate malaria-causing agent due to its characteristic quartan pattern of fever, in which clinical paroxysms occur at intervals of approximately 72 hours. This distinctive periodicity contrasts with the 48-hour erythrocytic cycle associated with tertian malaria, which is characteristic of P. vivax and is also frequently observed in infections caused by P. falciparum. P. malariae also produces fewer merozoites per schizont (approximately 6–12) and demonstrates a strict preference for mature red blood cells (RBCs) [43].

Quartan malaria results from infection with P. malariae, a species that occurs worldwide but displays a heterogeneous and often focal geographic distribution. Its prevalence is generally lower than that of P. falciparum and P. vivax. The parasite develops slowly within its mosquito host and is typically associated with relatively low parasite burdens in infected individuals. Because P. malariae lacks hypnozoites, it does not cause relapsing malaria. However, chronic low-level parasitemia can persist for extended periods, allowing infections to remain detectable for decades after the initial exposure [43].

The asexual erythrocytic cycle of P. malariae lasts approximately 72 hours, compared with 48 hours in most other human Plasmodium species. Erythrocytes infected with P. malariae typically remain normal in size throughout schizogony. The early ring stages may resemble those of other Plasmodium species, particularly P. vivax. As the parasite develops, it exhibits little amoeboid activity and often assumes a characteristic elongated band-like form that may extend across much of the host erythrocyte. Infected erythrocytes typically remain normal in size. In some cases, the erythrocyte cytoplasm contains fine stippling known as Ziemann’s dots, which are usually visible only in strongly stained preparations. The parasite also produces coarse, dark brown pigment [23].

During schizogony, 6–12 merozoites are formed, averaging approximately eight per schizont. These may occasionally be arranged in a characteristic rosette pattern, symmetrically surrounding a central pigment mass, although they are more often irregularly distributed in mature schizonts [41].

Gametocytes of P. malariae are difficult to distinguish from developing trophozoites, but when mature they are slightly larger than mature trophozoites and tend to be ovoid in shape. They contain more pigment than trophozoites at all stages. In thick blood films, trophozoites of P. malariae do not assume the swallow-like forms observed in some other species but usually appear as small chromatin dots associated with a round or slightly elongated mass of cytoplasm. Older trophozoites are compact, and prominent malarial pigment becomes the dominant morphological feature. A fully developed schizont containing approximately 8 merozoites is characteristic [36, 41].

4.5 Plasmodium knowlesi – Knowles and Das Gupta, 1932

Knowlesi malaria is caused by P. knowlesi, a zoonotic malaria parasite prevalent across Southeast Asia. Human infections may range from uncomplicated illness to severe disease with a risk of fatal outcomes. Epidemiological studies have identified Malaysia, Borneo, Myanmar, the Philippines, Singapore, and Thailand as the principal regions where transmission and reported case numbers are the highest [44]. This species was identified for the first time in 1931 in Macaca fascicularis – long-tailed macaques, in which it may cause fatal infection. P. knowlesi may also infect humans. In the past, P. knowlesi was used as a therapeutic tool for the treatment of neurosyphilis.

The first naturally acquired human infection with knowlesi malaria was described in 1965 in an American traveler returning from Malaysia [45, 46]. For many years, knowlesi malaria was frequently misdiagnosed as P. malariae infection because of the morphological similarities between the two species. The introduction of molecular diagnostic methods demonstrated that many of these infections were caused not by P. malariae, but by a distinct species, P. knowlesi.

Since 2004, the number of reported cases of knowlesi malaria has increased significantly. Analyses of archival samples have shown that P. knowlesi infections had already been occurring in Malaysia since the 1950s, with a prevalence of up to 70% in Sarawak [46].

P. knowlesi has a 24-hour asexual replication cycle, allowing large numbers of parasites to appear in the bloodstream within a relatively short period [46]. As a result, infection may progress rapidly and become severe or life-threatening.

Morphologically, the erythrocytic stages of P. knowlesi are like those of P. malariae and P. falciparum, which created obstacles in microscopic diagnostics before the era of PCR.

Hypnozoites have not been identified in this species, and true relapses are therefore not believed to occur. Known mosquito vectors of P. knowlesi include Anopheles cracens, An. latens, An. hackeri, and An. leucosphyrus.

Clinical symptoms of infection usually appear approximately 11 days after infection. The main manifestations include fever, chills, headache, rigors, and vomiting. Laboratory findings commonly include elevated C-reactive protein levels and thrombocytopenia. In some cases, severe complications such as acute respiratory distress syndrome and hepatorenal failure may develop and are associated with a mortality rate of approximately 2%.

Because P. knowlesi has a quotidian cycle, rapid diagnosis and prompt treatment are essential. If specific rapid diagnostic tests for knowlesi malaria are unavailable, patients who have recently traveled to Southeast Asia and present with infection microscopically diagnosed as P. malariae, accompanied by high parasitemia, should be treated urgently, as in cases of falciparum malaria [32, 46, 47].