MacroPath Logo
Search: for Help
About
Introduction
Statistics
Your PHIDIAS
Register or Login
Philert
Submission
Curated Data
Victors
BBP (Brucella)
Phinfo
Phinet
HazARD
Data Analysis
Phigen
Pacodom
BLAST
Help & Documents
Documents
FAQs
Links
Acknowledgements
Disclaimer
Contact Us
UMMS Logo

Table of Contents:
Taxonomy Information
  1. Species:
    1. Plasmodium falciparum (Website 1):
      1. Common Name: Malaria parasite P. falciparum
      2. GenBank Taxonomy No.: 5833
      3. Description: Malaria is the most serious tropical disease of humankind and the cause of much debilitation and morbidity in areas where it is endemic. Globally, the annual disease burden in over 90 endemic countries is estimated at 250 to 500 million clinical cases with the greatest impact in sub-Saharan Africa, where it claims the lives of 1.5 to 2 million children every year. The disease is caused by a protozoan parasite from the genus Plasmodium, transmitted through the bite of the female Anopheles mosquito. Four species of Plasmodium are known to cause disease in humans, P. vivax, P. ovale, P. malariae and P. falciparum, the latter being the most virulent and the major cause of mortality(de Souze and Riley, 2002).
      4. Variant(s):
        • Plasmodium falciparum (isolate 311) (Website 2):
        • Plasmodium falciparum (isolate 7G8) (Website 3):
          • GenBank Taxonomy No.: 57266
          • Parents: Plasmodium falciparum
          • Description: The parental stock of falciparum parasites to be cloned was isolated from 12 year-old male near Manaus, Brazil on 12 March 1980 and cryopreserved. The isolate IMTM22 was later put into a static culture system(Burkot et al., 1984). After more than 19 weeks in culture, a sample of IMTM 22 was cloned by the limiting dilution method (Burkot et al., 1984). The clones, of which 7G8 is one, were subcultured at weekly intervals until three flasks of each clone were established(Burkot et al., 1984).
        • Plasmodium falciparum (isolate CAMP / Malaysia) (Website 4):
        • Plasmodium falciparum (isolate CDC / Honduras) (Website 5):
          • GenBank Taxonomy No.: 5836
          • Parents: Plasmodium falciparum
          • Description: The Honduras I/CDC strain (isolated 25 Jan 1980, and cultured from frozen material in May and in June 1980) was placed in culture from frozen material on Sept 1981, and maintained by the continuous flow technique(Bhasin and Trager, 1984).
        • Plasmodium falciparum (isolate DD2) (Website 6):
        • Plasmodium falciparum (isolate FC27 / Papua New Guinea ) (Website 7):
          • GenBank Taxonomy No.: 5837
          • Parents: Plasmodium falciparum
          • Description: FCQ-27/PNG was collected originally from a 4 year old donor(Chen et al., 1980). Venous blood was collected asceptically into bottles containing heparin from 37 patients with clinical malaria in Madang Hospital, Madang Province, Papua New Guinea. The cells were washed twice in culture medium lacking serum and resuspended to 10% v/v in medium for transportation on wet ice by air to Brisbane or Melbourne where they were cultured after 1 to 4 days in transit (Chen et al., 1980).
        • Plasmodium falciparum (isolate FcB1 / Columbia) (Website 8):
        • Plasmodium falciparum (isolate FCBR / Columbia) (Website 9):
          • GenBank Taxonomy No.: 33631
          • Parents: Plasmodium falciparum
          • Description: Two chloroquine-resistant strains were used to infect owl monkeys or squirrel monkeys: the culture-adapted FCBR (FCB) strain, originally isolated from a patient in Bogota, Colombia, and the Palo Alto strain, originally isolated from a patient in Uganda and then adapted to owl monkeys(Raether et al., 1989).
        • Plasmodium falciparum (isolate FCH-5) (Website 10):
        • Plasmodium falciparum (isolate FCM17 / Senegal) (Website 11):
          • GenBank Taxonomy No.: 5845
          • Parents: Plasmodium falciparum
          • Description: Malaria parasites from three different strains (FCM-17 from Senegal, FCM-22 from Madagascar, and FCM-29 from Cameroon) were cultured in vitro(Lenstra et al., 1987).
        • Plasmodium falciparum (isolate FCR-3 / Gambia) (Website 12):
          • GenBank Taxonomy No.: 5838
          • Parents: Plasmodium falciparum
          • Description: This strain originated from the Gambia, West Africa. A culture line was established in 1976 at the Rockefeller Institute directly from an infected patient(Fandeur et al., 1991).
        • Plasmodium falciparum (isolate fid3 / India) (Website 13):
          • GenBank Taxonomy No.: 70152
          • Parents: Plasmodium falciparum
          • Description: Blood samples were collected from three malaria patients in different geographic areas of India. These locations (New Delhi, Shahjahanpur and Jabalpur) are separated by a few hundred kilometers from each other. These isolates, FID3, FIS1 and FIJ4, were maintained in continuous in vitro culture using the standard candle-jar method of Trager and Jensen(Bhattacharya et al., 1995).
        • Plasmodium falciparum (isolate hb3) (Website 13a):
          • GenBank Taxonomy No.: 137071
          • Parents: Plasmodium falciparum
          • Description: Three clones have been prepared from the Honduras I/CDC strain of Plasmodium falciparum by a method of microscopic selection. One of these (HB-2) does not form gametocytes whereas the others (HB-1 and HB-3) do. All three are as resistant to pyrimethamine as the original line, and all three form knobs on the erythrocyte surface(Bhasin and Trager, 1984).
        • Plasmodium falciparum (isolate IMR143) (Website 14):
          • GenBank Taxonomy No.: 57268
          • Parents: Plasmodium falciparum
          • Description: Isolates FCQ27/PNG (FC27), IMR143/PNG (IMR143), IMR144/PNG (IMR144), IMR147/PNG (IMR147) and MAD71/PNG (Mad71) were all obtained from Papua New Guineans living in the Madang Province of Papua New Guinea(Saint et al., 1987).
        • Plasmodium falciparum (isolate K1 / Thailand) (Website 15):
        • Plasmodium falciparum (isolate KF1916) (Website 16):
        • Plasmodium falciparum (isolate LE5) (Website 17):
          • GenBank Taxonomy No.: 5840
          • Parents: Plasmodium falciparum
          • Description: This strain was isolated from a native Liberian by Dr. M. Swartz and sent to us on 21 October, 1980. It was cryopreserved on the day of arrival. A vial was subsequently thawed and the strain was grown for 16 days before cloning(Graves et al., 1984). Parasites in rapid growth phase were cloned by limiting dilution in 96-well plates or tissue-culture flasks(Graves et al., 1984). Strain LE5 was one of these clones(Graves et al., 1984).
        • Plasmodium falciparum (isolate mad20 / Papua New Guinea) (Website 18):
        • Plasmodium falciparum (isolate mad71 / Papua New Guinea) (Website 19):
          • GenBank Taxonomy No.: 70154
          • Parents: Plasmodium falciparum
          • Description: Isolates FCQ27/PNG (FC27), IMR143/PNG (IMR143), IMR144/PNG (IMR144), IMR147/PNG (IMR147) and MAD71/PNG (Mad71) were all obtained from Papua New Guineans living in the Madang Province of Papua New Guinea(Saint et al., 1987).
        • Plasmodium falciparum (isolate NF54) (Website 20):
          • GenBank Taxonomy No.: 5843
          • Parents: Plasmodium falciparum
          • Description: Isolates of P. falciparum were obtained from malaria imported into the Netherlands. Most of the patients were infected during their visit to different parts of Africa(Ponnudurai et al., 1981). The origin of NF54 is listed as Schiphol, the Amsterdam airport(Ponnudurai et al., 1981).
        • Plasmodium falciparum (isolate NF7 / Ghana) (Website 21):
        • Plasmodium falciparum (isolate nig32 / Nigeria) (Website 22):
        • Plasmodium falciparum (isolate PALO ALTO / UGANDA) (Website 23):
          • GenBank Taxonomy No.: 57270
          • Parents: Plasmodium falciparum
          • Description: The Uganda-Palo Alto (FUP) strain of Plasmodium falciparum was originally isolated from a patient who had contracted the infection in Uganda and was hospitalized at Stanford Medical Center, Palo Alto, California, in 1966. In 1967, blood-induced infections with this isolate were established in Aotus trivirgatus monkeys at Stanford. The FUP strain was maintained by serial passage in Aotus monkeys by Dr. Schmidt of the Southern Research Institute, Birmingham, Alabama. In 1970, the FUP monkey-passaged strain was obtained from Dr. Schmidt and maintained in this laboratory at the University of Hawaii by serial passage in Aotus monkeys. In 1977, continuous cultures of the FUP strain in human erythrocytes were established at the University of Hawaii and have been maintained in this laboratory since that time(Chang et al., 1988).
        • Plasmodium falciparum (isolate RO-33 / Ghana) (Website 24):
        • Plasmodium falciparum (isolate T4 / Thailand) (Website 25):
        • Plasmodium falciparum (isolate TAK 9) (Website 26):
          • GenBank Taxonomy No.: 57276
          • Parents: Plasmodium falciparum
          • Description: The isolate used (T9) was obtained from a patient at Tak, Thailand, and was established in continuous culture by Sodsri Thaithong at Chulalongkorn University, Bangkok, by the petri-dish method(Rosario, 1981).
        • Plasmodium falciparum (isolate thtn / thailand) (Website 27):
        • Plasmodium falciparum (isolate V1) (Website 28):
          • GenBank Taxonomy No.: 5847
          • Parents: Plasmodium falciparum
          • Description: Various isolates of Plasmodium falciparum originally obtained from patients attending outpatient clinics in hospitals either in the respective countries of origin of the isolate or in the United States were cryopreserved and later thawed and cultured continuously(Udeinya et al., 1983). V1 was obtained from a Vietnamese refugee in Canada who traveled through Cambodia on his way to Canada. The origin of the isolate is either Vietnam or Cambodia. The date of isolation is 3 December 1980(Udeinya et al., 1983).
        • Plasmodium falciparum (isolate WELLCOME) (Website 29):
        • Plasmodium falciparum 3D7 (Website 30):
          • GenBank Taxonomy No.: 36329
          • Parents: Plasmodium falciparum
          • Description: 3D7 is a clone from NF54 which was isolated from a patient who lived near the airport in Amsterdam. The origin of the infection is unknown(Miller et al., 1993).
Lifecycle Information
  1. Exo-erythrocytic cycle
    1. Stage Information:
      1. Sporozoite:
        • Size: 11 um in length and 1.0 um in diameter.
        • Shape: Elongate
        • Description: The sporozoites are more elongated than either erythrocytic or exoerythrocytic merozoites. They measure about 11 um in length and 1.0 um in diameter. The organelles found in the sporozoites are essentially the same as those of the merozoites(Aikawa and Seed, 1980).
      2. Trophozoite:
        • Shape: Irregular
        • Picture(s):
          • Trophozoite Progression (Website 125)



            Description: Fig 1: Normal red cells. Figs. 2-10: Increasingly mature ring stage parasties. Illustartion from Coatneyu GR, Collins WE, Warren M, Contacos PG. The Primate Malarias. U.S. Department of Health, Education and Welfare, Bethesda, 1971. Copyright CDC(Website 125).
        • Description: The trophozoite is the stage in which ingestion of host cell cytoplasm and growth of the parasite occur(Aikawa and Seed, 1980). The trophozoite is surrounded by only a single plasma membrane in addition to the parasitophorous vacuole membrane which originated from the host cell. Apical end organelles such as polar rings, rhoptries, micronemes, and spherical bodies also break down. The trophozoite becomes irregular in shape(Aikawa and Seed, 1980).
      3. Schizont:
        • Shape: Round
        • Picture(s):
        • Description: The schizont is defined as a parasite possessing more than one nucleus. The schizont is larger than either the merozoite or uninucleate trophozoite. During schizogony, nuclear division and differentiation of the cytoplasmic organelles occur(Aikawa and Seed, 1980). With the progression of merozoite budding, various organelles, including a nucleus, mitochondria, spherical bodies together with endoplasmic reticulum, and ribosomes migrate into the developing merozoites from the schizont. As the merozoites develop and grow, the size of the original schizont decreases until finally only a residual body with malarial pigment particles remains(Aikawa and Seed, 1980).
      4. Merozoite:
        • Size: The exoerythrocytic merozoite is more elongate than the erythrocytic merozoite and is 3 to 4 um in length and 1 to 2 um in width.
        • Shape: The merozoite is oval in shape
        • Description: The exoerythrocytic merozoite is more elongate than the erythrocytic merozoite and is 3 to 4 um in length and 1 to 2 um in width. There are more micronemes in the exoerythrocytic stages, and the rhoptries appear to be more elongated(Aikawa and Seed, 1980). Merozoites can be seen by light microscopy to round up upon entry into a new red blood cell. This phenomenon has been observed in detail by electron microscopy. The round-up process appears to be caused by the rapid degradation of the inner membrane and microtubules of the pellicular complex(Aikawa and Seed, 1980). The free merozoite is very small, approximately 1.2 um long, but it contains all things necessary to invade and establish itself in a new RBC. At the apex of the egg-shaped merozoite are three sets of secretory vesicles: (1) the twin pear-shaped rhoptries; (2) the more numerous but smaller micronemes; and (3) small rounded vesicles called dense granules. The nucleus lies at the other end, and a plastid and a mitochondrion lie along one side of the merozoite, near a band of two or three microtubules. Apically, three dense cytoskeletal rings (polar rings) brace the apical prominence. A flat sac of membrane underlies most of the merozoite surface membrane, forming with it the merozoite's pellicle, which lines the whole cell except most apically. The merozoite also contains numerous free ribosomes. Over the whole surface of the merozoite, there is a thick, bristly adhesive coat(Bannister and Mitchell, 2003).
    2. Progression Information:
      1. Sporozoite-Trophozoite:
        • From Stage: Sporozoite
        • To Stage: Trophozoite
        • Description: In their search for capillaries, mosquitoes probe the skin of the host and, in the process, deposit sporozoites. Sporozoites remain in the skin for more than 5 mins and it is not clear how they enter the bloodstream(Kappe et al., 2003). When injected directly into the bloodstream, sporozoites invade hepatocytes within a few minutes. Most circulating sporozoites are sequestered in the liver after a single passage, suggesting that a specific receptor is present on the cells lining the sinusoids(Kappe et al., 2003). Once within a hepatic cell, the parasite metamorphoses into a feeding trophozoite(Schmidt and Roberts, 1985).
      2. Trophozoite-Schizont:
        • From Stage: Trophozoite
        • To Stage: Schizont
        • Description: After about a week the trophozoite is mature and begins schizogony. Numerous daughter nuclei are first formed, transforming the parasite into a schizont, also known as a cryptozoite(Schmidt and Roberts, 1985).
      3. Schizont-Merozoite:
        • From Stage: Schizont
        • To Stage: Merozoite
        • Picture(s):
          • Rupturing Schizont (Website 128)



            Description: Ruptured schizont in a thin blood smear. Copyright CDC(Website 128).
        • Description: During the nuclear divisions the nuclear membranes persist, and the microtubular spindle fibers are formed within the nucleus. The mitochondrion becomes larger during the growth of the trophozoite, forms buds, and then breaks up into many mitochondria. Elements of the apical complex form subjacent to the outer membrane, and schizogony proceeds. The merozoites thus formed after cytokinesis are referred to as metacryptozoites(Schmidt and Roberts, 1985). Eventually, merozoites leave liver cells to penetrate erythrocytes in the blood, initiating the erythrocytic cycle(Schmidt and Roberts, 1985).
    3. Picture(s):
      • Malaria Life Cycle (Website 124)



        Description: The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host (1). Soporozoites infect liver cells and mature into schizonts (2), which rupture and release merozoites (3). Of note, in P. vivax and P. ovale a dormant stage, hynozoites, can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later (4). After this initial replication in the liver, called(exo-erythrocytic schizogony (A), the parasites undergo asexual multiplication in the erythrocytes, called erythrocytic schizogony (B). Merozoites infect red blood cells (5). The ring stage trophozoites mature into schizonts, which rupture releasing merozoites (6). Some parasites differentiate into sexual erythrocytic stages (gametocytes) (7). 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 (8). The parasites' multiplication in the mosquito is known as the sporogonic cycle (C). While in the mosquito's stomach, the microgametes penetrate the macrogametes generation zygotes (9). The zygotes in turn become motile and elongated ookinetes (10) which invade the midgut wall of the mosquito where they develop into oocysts (11). The oocysts grow, rupture, and release sporozoites (12), which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle (1). Copyright CDC(Website 124).
    4. Description: After being injected into the bloodstream, the sporozoites quickly disappear (within an hour) from the circulating blood. Their immediate fate was a great mystery until the mid-1940's, when it was shown that within 1 or 2 days they enter the parenchyma of the liver or other internal organ, depending on the species of Plasmodium. Where they are the first 24 hours still is unknown. Entry into the liver initiates a series of asexual reproductions known as the preerythrocytic cycle or primary exoerythrocytic schizogony, often abbreviated as the PE or EE stage. Once within a hepatic cell, the parasite metamorphoses into a feeding trophozoite(Schmidt and Roberts, 1985). The preerythrocytic stages of mammalian malaria parasites apparently undergo within hepatic cells only a single developmental cycle that forms many merozoites that invade erythrocytes(Trager, 1986).
  2. Erythrocytic Cycle
    1. Stage Information:
      1. Trophozoite:
        • Size: The young ring forms of P. falciparum, as usually seen in the peripheral blood, are very small, measuring about one-sixth of the diameter of a red blood cell.. Later in the attack, the ring forms of P. falciparum may be considerably larger, measuring one-quarter and sometimes nearly one-half the diameter of a red cell, and may sometimes be mistaken for parasites of P. malariae.
        • Shape: Irregular
        • Picture(s):
        • Description: The trophozoite is surrounded by only a single plasma membrane in addition to the parasitophorous vacuole membrane which originated from the host cell. Apical end organelles such as polar rings, rhoptries, micronemes, and spherical bodies also break down. The trophozoite becomes irregular in shape(Aikawa and Seed, 1980). This is the period of most active feeding, growth and RBC modification. New molecules are exported into the RBC, some assembling into flat membranous sacs of various forms, including those visible in stained smears as Maurer's clefts. Others interact with the RBC membrane and cytoskeleton to form small knobs on its surface, and some penetrate it, for example, P. falciparum erythrocyte membrane protein (PfEMP)1 to stick the infected RBC to the endothelium of blood vessels, thus reducing parasite removal from the blood stream by the defenses of the body via the spleen. If the infected RBC adheres to brain-blood vessel walls, cerebral malaria can result, while in the placenta, fetal growth can be affected by similar cytoadherence. Other exported molecules increase RBC permeability to nutrients. The parasite continues feeding on haemoglobin, and the haeme products of haemoglobin digestion crystallize into particles of dark pigment, haemozoin, scattered within the food (pigment) vacuole(Bannister and Mitchell, 2003).
      2. Schizont:
        • Size: Smaller than normal red cells.. When the schizont is fully grown it occupies about two-thirds of the red cell.
        • Picture(s):
        • Description: The schizont is defined as a parasite possessing more than one nucleus. The schizont is larger than either the merozoite or uninucleate trophozoite. During schizogony, nuclear division and differentiation of the cytoplasmic organelles occur(Aikawa and Seed, 1980). With the progression of merozoite budding, various organelles, including a nucleus, mitochondria, spherical bodies together with endoplasmic reticulum, and ribosomes migrate into the developing merozoites from the schizont. As the merozoites develop and grow, the size of the original schizont decreases until finally only a residual body with malarial pigment particle remains(Aikawa and Seed, 1980).
      3. Merozoite:
        • Size: 1.5 um in length, and 1 um in diameter.
        • Shape: The merozoite is oval in shape.
        • Description: With the progression of merozoite budding, various organelles, including a nucleus, mitochondria, spherical bodies together with endoplasmic reticulum, and ribosomes migrate into the developing merozoites from the schizont. As the merozoites develop and grow, the size of the original schizont decreases until finally only a residual body with malarial pigment particles remains(Aikawa and Seed, 1980). The merozoite is oval in shape, 1.5 um in length, and 1 um in diameter, posses a nucleus and various cytoplasmic organelles, and is bounded by a pellicular complex(Aikawa and Seed, 1980).
      4. Microgametocyte:
        • Shape: The gametocytes of Plasmodium falciparum are sausage-shaped, though they are generally referred to as crescents. They are totally unlike the sexual stages of any other human Plasmodium. The male cell is shorter and more blunt at the ends than is the female, and has more dispersed chromatin and pigment.
        • Description: When development of the merozoites is complete, the host cell ruptures(Schmidt and Roberts, 1985). After an indeterminate number of asexual generations, some merozoites enter erythrocytes to become macrogamonts (macrogametocytes) and microgamonts (microgametocytes). The size and shape of these cells are characteristic for each species. Unless they are ingested by a mosquito, gametocytes soon die and are phagocytized by the reticuloendothelial system(Schmidt and Roberts, 1985).
      5. Macrogametocyte:
        • Shape: The gametocytes of Plasmodium falciparum are sausage-shaped, though they are generally referred to as crescents. They are totally unlike the sexual stages of any other human Plasmodium.
        • Description: When development of the merozoites is complete, the host cell ruptures(Schmidt and Roberts, 1985). After an indeterminate number of asexual generations, some merozoites enter erythrocytes to become macrogamonts (macrogametocytes) and microgamonts (microgametocytes). The size and shape of these cells are characteristic for each species. Unless they are ingested by a mosquito, gametocytes soon die and are phagocytized by the reticuloendothelial system(Schmidt and Roberts, 1985).
    2. Progression Information:
      1. Merozoite-Trophozoite:
        • From Stage: Merozoite
        • To Stage: Trophozoite
        • Description: The 48-h P. falciparum intraerythrocytic developmental cycle (IDC) initiates with merozoite invasion of red blood cells (RBCs) and is followed by the formation of the parasitophorous vacuole (PV) during the ring stage. The parasite then enters a highly metabolic maturation phase, the trophozoite stage, prior to parasite replication(Bozdech et al., 2003). The IDC represents all of the stages in the development of P. falciparum responsible for the symptoms of malaria and is also the target for the vast majority of antimalarial drugs and vaccine strategies(Bozdech et al., 2003). On entry into an erythrocyte, the merozoite again transforms into a trophozoite. The host cytoplasm ingested by the trophozoite forms a large food vacuole, giving the young plasmodium the appearance of a ring of cytoplasm with the nucleus conspicuously displayed at one edge(Schmidt and Roberts, 1985).
      2. Trophozoite-Schizont:
        • From Stage: Trophozoite
        • To Stage: Schizont
        • Description: The parasite rapidly develops into a schizont. The stage in the erythrocytic schizogony at which the cytoplasm is coalescing around the individual nuclei, before cytokinesis, is called the segmenter(Schmidt and Roberts, 1985).
      3. Schizont-Merozoite:
        • From Stage: Schizont
        • To Stage: Merozoite
        • Description: Within the human bloodstream, the malaria organism Plasmodium falciparum is an intracellular parasite, cyclically invading, feeding and multiplying in red blood cells (RBCs). In its multiplicative phase (the schizont), the parasite generates in the region of 16 invasive forms (merozoites) that bud off from its perimeter and are eventually released from their surrounding membranes to invade fresh RBCs. During this phase the nucleus undergoes four or more mitotic divisions, whereas, in the cytoplasm, there ensues the coordinated synthesis, targeting and assembly of invasion-specific organelles. These include three types of secretory vesicle: rhoptries, micronemes and dense granules, which have different shapes, sizes, contents and roles in invasion(Bannister et al., 2003). When development of the merozoites is completed, the host cell ruptures, releasing parasite metabolic wastes and residual body, including hemozoin. The metabolic wastes thus released are one factor responsible for the characteristic symptoms of malaria, although hemozoin itself is nontoxic(Schmidt and Roberts, 1985).
      4. Merozoite-Microgametocyte_Macrogametocyte:
        • From Stage: Merozoite
        • To Stage: Microgametocyte, Macrogametocyte
        • Description: Concomitantly, a small proportion of the parasites terminally differentiate from merozoites into gametocytes. Little is known about the genes that control the developmental switch to the gametocyte stage. This is a subject of key importance, because inhibition of gametocyte differentiation could be powerful means for the control of malaria transmission(Ghosh et al., 2000). After an indeterminate number of asexual generations, some merozoites enter erythrocytes and become macrogamonts (macrogametocytes) and microgamonts (microgametocytes). The size and shape of these cells are characteristic for each species; they also contain hemozoin. Unless they are ingested by a mosquito, gametocytes soon die and are phagocytized by the reticuloendothelial system(Schmidt and Roberts, 1985).
    3. Picture(s):
      • Malaria Life Cycle (Website 124)



        Description: The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host (1). Soporozoites infect liver cells and mature into schizonts (2), which rupture and release merozoites (3). Of note, in P. vivax and P. ovale a dormant stage, hynozoites, can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later (4). After this initial replication in the liver, called(exo-erythrocytic schizogony (A), the parasites undergo asexual multiplication in the erythrocytes, called erythrocytic schizogony (B). Merozoites infect red blood cells (5). The ring stage trophozoites mature into schizonts, which rupture releasing merozoites (6). Some parasites differentiate into sexual erythrocytic stages (gametocytes) (7). 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 (8). The parasites' multiplication in the mosquito is known as the sporogonic cycle (C). While in the mosquito's stomach, the microgametes penetrate the macrogametes generation zygotes (9). The zygotes in turn become motile and elongated ookinetes (10) which invade the midgut wall of the mosquito where they develop into oocysts (11). The oocysts grow, rupture, and release sporozoites (12), which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle (1). Copyright CDC(Website 124).
    4. Description: Malarial parasites have a life cycle even more complicated than that of trypanosomes and not yet fully reproduced in culture. In their vertebrate hosts (lizards, birds, or mammals), the principal propagative cycle, and the one responsible for the human disease malaria, occurs intracellularly within the red blood cells. This is a cycle of asexual reproduction in which a small invasive form, a merozoite, enters an erythrocyte, grows rapidly within it, and then undergoes schizogony to produce 8 to 24 daughter merozoites (depending upon the species), each potentially able to invade another erythrocyte and reproduce the cycle. Each cycle takes 24, 48, or 72 hr, again depending on the species. This portion of the life cycle clearly is capable of indefinite propagation as long as the parasites are supplied with appropriate red cells and other environmental conditions(Trager, 1986).
  3. Sporogonic Cycle
    1. Stage Information:
      1. Microgametocyte:
        • Shape: The gametocytes of Plasmodium falciparum are sausage-shaped, though they are generally referred to as crescents. They are totally unlike the sexual stages of any other human Plasmodium. The male cell is shorter and more blunt at the ends than is the female, and has more dispersed chromatin and pigment.
        • Description: After an indeterminate number of asexual generations, some merozoites enter erythrocytes to become macrogamonts (macrogametocytes) and microgamonts (microgametocytes). The size and shape of these cells are characteristic for each species. Unless they are ingested by a mosquito, gametocytes soon die and are phagocytized by the reticuloendothelial system(Schmidt and Roberts, 1985).
      2. Macrogametocyte:
        • Shape: The gametocytes of Plasmodium falciparum are sausage-shaped, though they are generally referred to as crescents. They are totally unlike the sexual stages of any other human Plasmodium.
        • Picture(s):
          • Mature Macrogametocytes (Website 131)



            Description: Figs. 27, 28: Mature macrogametocytes (female); Figs. 29, 30: Mature microgametocytes (male)Illustration from: Coatney GR, Collins WE, Warren M, Contacos PG. The Primate Malarias. U.S. Department of Health, Education and Welfare, Bethesda, 1971. Copyright CDC(Website 131).
        • Description: After an indeterminate number of asexual generations, some merozoites enter erythrocytes to become macrogamonts (macrogametocytes) and microgamonts (microgametocytes). The size and shape of these cells are characteristic for each species. Unless they are ingested by a mosquito, gametocytes soon die and are phagocytized by the reticuloendothelial system(Schmidt and Roberts, 1985).
      3. Microgamete:
        • Size: 16-25 um in length
        • Picture(s):
          • Gametocytes in Thick Blood Smear (Website 131)



            Description: Two gametocytes captured from a thick blood smear. Copyright CDC(Website 131).
        • Description: Four to six microgametes are produced during exflagellation, each measuring 16-25 um in length(Aikawa and Seed, 1980).
      4. Macrogamete:
        • Shape: The crescent-shaped macrogamete rounds up as it sheds the host red cell.
        • Description: As the microgametocyte becomes extracellular, within 10 to 12 minutes its nucleus divides repeatedly to form six to eight daughter nuclei, each of which is associated with elements of a developing axoneme. The doubled outer membrane of the microgametocyte becomes interrupted; the flagellar buds with their associated nuclei move peripherally between the interruptions and then continue outward covered by the outer membrane of the gametocyte. These break free and are the microgametes(Schmidt and Roberts, 1985).
      5. Zygote:
        • Shape: When cultured for 10 h after exflagellation, most zygotes showed an oval or budding shape, indicating they had just started transformation into the invasive form.
        • Description: The microgamete swims about until it finds a macrogamete, which it penetrates and fertilizes. The resultant diploid zygote quickly elongates to become a motile ookinete(Schmidt and Roberts, 1985).
      6. Ookinete:
        • Size: 11-13 um in length.
        • Shape: The ookinete of P. falciparum is generally more slender than that of P. vivax and measures 11-13 um in length.
        • Description: The ookinete of P. falciparum is generally more slender than that of P. vivax and measures 11-13 um in length. In typical fashion, one end of the ookinete is truncated and the other rounded and more bulbous. The two poles appear light yellow and pink, respectively, in Giemsa-stained preparations. The nucleus is irregular in shape and slightly acentric. The cytoplasm is uneven, appearing pigmented and at times striated because of the presence of the anterior organelles(Aikawa and Seed, 1980).
      7. Oocyst:
        • Size: 8 to 60 um
        • Description: Oocyst development requires approximately 10 days. This varies, however, depending on the species of anopheline mosquito infected and on an assortment of environmental conditions. At maturity, oocyst diameters range from 8 to 60 um. The distribution of coarse, black pigment granules (10 to 20), in semicircular rows or chains, is somewhat characteristic of the species. The processes of nuclear division and cytoplasmic segmentation into sporoblastic islands are morphologically similar to those of other species(Aikawa and Seed, 1980).
      8. Sporozoite:
        • Size: 11 um in length and 1.0 um in diameter.
        • Shape: Elongate
        • Description: The sporozoites are more elongated than either erythrocytic or exoerythrocytic merozoites. They measure about 11 um in length and 1.0 um in diameter. The organelles found in the sporozoites are essentially the same as those of the merozoites(Aikawa and Seed, 1980).
    2. Progression Information:
      1. Microgametocyte-Microgamete:
        • From Stage: Microgametocyte
        • To Stage: Microgamete
        • Description: Once the parasites have entered the mosquito gut, they differentiate from gametocytes into gametes. Each male gametocyte generates eight haploid motile gametes within minutes of a bloodmeal by a process known as exflagellation. Exflagellation can be induced in vitro by dropping the temperature of cultured gametes from 37 C to 28 C, thus stimulating the change from host to vector temperatures, and by either increasing the pH of cultured gametes from 7.5 to 8.2 or by the addition of a gametocyte-activating factor (GAF). GAF has been isolated from the mosquito gut and has been shown to stimulate exflagellation in vitro. GAF has recently been identified as zanthurenic acid, and intermediate product of tryptophan metabolism. It is not clear what role, if any zanthurenic acid or pH play in triggering exflagellation in vivo, and neither is their mechanism of action known(Ghosh et al., 2000). The microgametocyte displays a rather astonishing transformation, exflagellation. As the microgametocyte becomes extracellular, within 10 to 12 minutes its nucleus divides repeatedly to form six to eight daughter nuclei, each of which is associated with the elements of a developing axoneme. The doubled outer membrane of the microgametocyte becomes interrupted; the flagellar buds with their associated nuclei move peripherally between the interruptions and then continue outward covered by the outer membrane of the gametocyte. These break free and are the microgametes. The stimulus for exflagellation is an increase in pH caused by escape of dissolved carbon dioxide from the blood. The life span of the microgametes is short, since they contain little more than the nuclear chromatin and the flagellum covered by a membrane(Schmidt and Roberts, 1985).
      2. Macrogametocyte-Macrogamete:
        • From Stage: Macrogametocyte
        • To Stage: Macrogamete
        • Description: After release from its enclosing erythrocyte, maturation of the macrogametocyte to the macrogamete involves little obvious change other than a shift of the nucleus toward the periphery(Schmidt and Roberts, 1985).
      3. Macrogamete_Microgamete-Zygote:
      4. Zygote-Ookinete:
        • From Stage: Zygote
        • To Stage: Ookinete
        • Description: The diploid zygote quickly elongates to become a motile ookinete(Schmidt and Roberts, 1985). After fertilization, the resulting zygotes transform into motile ookinetes. Little is known about the factors that govern this dramatic morphological transition. Comparison of the number of gametocytes ingested by the mosquito with the number of resulting ookinetes indicates that only a small proportion of the parasites complete the differentiation from gametocyte to ookinete(Ghosh et al., 2000). Within approximately 24 hours of differentiation into an ookinete, the parasite has to cross two barriers, the peritrophic membrane and the midgut epithelium. How the ookinetes find their way from the interior of the blood mass out towards the epithelium (random or directed movement?) is an interesting question for which no information is available(Ghosh et al., 2000).
      5. Ookinete-Oocyst:
        • From Stage: Ookinete
        • To Stage: Oocyst
        • Description: The ookinete penetrates the peritrophic membrane in the mosquito's gut, migrates to the hemocoel side of the gut, and begins its transformation into an oocyst(Schmidt and Roberts, 1985). After traversing the midgut epithelium, the ookinete reaches the extracellular space between the midgut epithelium and the overlaying basal lamina where it develops into an oocyst(Ghosh et al., 2000).
      6. Oocyst-Sporozoite:
        • From Stage: Oocyst
        • To Stage: Sporozoite
        • Description: The oocyst progresses through a complex developmental process that, after 10 to 24 days (depending on species), culminates with the release of thousands of sporozoites into the hemocoel(Ghosh et al., 2000). As the oocyst differentiates, nuclei divide extensively in a common cytoplasm to yield several thousand sporozoites. One gene that is expressed selectively during this process encodes the circumsporozoite (CS) protein, a major sporozoite surface protein(Ghosh et al., 2000). Once in the hemocoel, sporozoites have access to a large number of organs and cell types. However, they invade only cells of the salivary gland epithelium(Ghosh et al., 2000). After salivary gland cell invasion, the parasites remain in the cytoplasm only transiently. They soon exit though the opposite (basal) side of the cell into the secretory cavity. Here, large numbers of sporozoites become organized in bundles and remain viable for the life of the mosquito. At each feeding cycle, a small number of sporozoites penetrate the secretory duct, from where they are expelled into the vertebrate host at the time of bloodfeeding(Ghosh et al., 2000).
    3. Picture(s):
      • Malaria Life Cycle (Website 124)



        Description: The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host (1). Soporozoites infect liver cells and mature into schizonts (2), which rupture and release merozoites (3). Of note, in P. vivax and P. ovale a dormant stage, hynozoites, can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later (4). After this initial replication in the liver, called(exo-erythrocytic schizogony (A), the parasites undergo asexual multiplication in the erythrocytes, called erythrocytic schizogony (B). Merozoites infect red blood cells (5). The ring stage trophozoites mature into schizonts, which rupture releasing merozoites (6). Some parasites differentiate into sexual erythrocytic stages (gametocytes) (7). 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 (8). The parasites' multiplication in the mosquito is known as the sporogonic cycle (C). While in the mosquito's stomach, the microgametes penetrate the macrogametes generation zygotes (9). The zygotes in turn become motile and elongated ookinetes (10) which invade the midgut wall of the mosquito where they develop into oocysts (11). The oocysts grow, rupture, and release sporozoites (12), which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle (1). Copyright CDC(Website 124).
    4. Description: The cycle of malaria in the vector mosquito is a self-limited one that begins with zygote formation by male and female gametes and ends with the formation of sporozoites(Trager, 1986).
Genome Summary
  1. Genome of Plasmodium falciparum
    1. Description: The P. falciparum 3D7 nuclear genome is composed of 22.8 megabases (Mb) distributed among 14 chromosomes ranging in size from approximately 0.643 to 3.29 Mb. Thus the P. falciparum genome is almost twice the size of the genome of the fission yeast Schizosaccharomyces pombe. The overall (A + T) composition is 80.6%, and rises to 90% in introns and intergenic regions. The structures of protein-encoding genes were predicted using several gene-finding programs and manually curated. Approximately 5,300 protein-encoding genes were identified, about the same as in S. pombe. This suggests an average gene density in P. falciparum of 1 gene per 4,338 base pairs (bp), slightly higher than was found previously with chromosomes 2 and 3 (1 per 4,500 bp and 1 per 4,800 bp, respectively). The higher gene density reported here is probably the result of improved gene finding software and larger training sets that enabled the detection of genes overlooked previously. Introns were predicted in 54% of P. falciparum genes, a proportion roughly similar to that in S. pombe and Dictyostelium discoideum, but much higher than observed in Saccharomyces cerevisiae where only 5% of genes contain introns. Excluding introns, the mean length of P. falciparum genes was 2.3 kb, substantially larger than in the other organisms in which the average gene lengths range from 1.3 to 1.6 kb. Plasmodium falciparum genes showed a markedly greater proportion of genes (15.5%) longer than 4 kb compared to S. pombe and S. cerevisiae (3.0% and 3.6%, respectively). The explanation for the increased gene length in P. falciparum is not clear. Many of these large genes encode uncharacterized proteins that may be cytosolic proteins, as they do not possess recognizable signal peptides. No transposable elements or retrotransposons were identified(Gardner et al., 2002B). Contiguous DNA sequences (contigs) have been obtained for chromosomes 1, 3, 4, 5 and 9, whereas chromosomes 6, 7, 8 and 13 contain a few gaps; most contigs have been ordered and oriented(Hall et al., 2002). As we were unable to produce unbroken sequence from telomere to telomere for all nine chromosomes, contiguous 'pseudo-chromosomes' were constructed by artificially joining all contigs that could be mapped to an individual chromosome. In most cases, the order and orientation of the contigs could be inferred using mapping data or read-pair information. Small contigs (of less than 5 kilobases, kb) that could not be mapped onto a chromosome have not been included in the analysis, and thus a small number of genes on the unmapped contigs will be missing from the genome sequence. The construction of pseudo-chromosomes does, however, have the advantage of allowing a global analysis of chromosome structure, and also removes redundancy from the analysis that would otherwise occur owing to contamination between chromosomes during purification and aberrant contigs formed during assembly(Hall et al., 2002). P. falciparum chromosomes were resolved on preparative pulsed field gels, and used to prepare shotgun libraries of 1 to 2 kilobase (kb) DNA fragments in plasmid vectors. Sequences of randomly selected clones were assembled, and gaps were closed using primer walking on plasmid templates or polymerase chain reaction (PCR) products. The cross-contamination of the chromosomal libraries with sequences from other chromosomes (up to 25%) and the high (A + T) content (80.6%) of P. falciparum DNA caused extreme difficulties in the gap closure process. Intergenic regions and introns frequently contained long runs of up to 50 consecutive A or T residues that were difficult to clone and sequence. The high (A plus T) content of the chromosomes also prevented the construction of large insert libraries that could be used to construct scaffolds of ordered and oriented contiguous DNA sequences (contigs) during assembly. Similar but more severe problems were reported in the sequencing of the (A plus T)-rich chromosome 2 of the slime mould Dictyostelium discoideum, illustrating the need to develop better methods for the cloning and sequencing of very (A plus T)-rich genomes. The reported sequences contain three or four short gaps (less than 2 kb) in each chromosome. Contigs comprising these chromosomes were joined end-to-end before annotation. Efforts to close the remaining gaps will continue.Examination of the sequences of chromosomes 2, 10, 11 and 14 revealed that the structure of these chromosomes was similar to that of the other chromosomes. All contained the 97 to 99% (A plus T) putative centromeric sequences reported previously. Conserved subtelomeric sequences were observed in chromosomes 2, 10 and 11, but most of these elements had been deleted from both ends of chromosome 14(Gardner et al., 2002). P. falciparum chromosome 12 is composed of 2,271,477 base pairs (bp). The sequence is completely contiguous; there are no gaps. This sequence is supported by a total of 91,191 reads (14.9-fold chromosome 12 coverage). Overall, the guanine-plus-cytosine (G plus C) content of chromosome 12 is 19.3%. As expected from this very low (G plus C) content, the P. falciparum chromosome 12 sequence contains many long runs of consecutive adenine and thymine residues. Runs of, at least, 20 such bases cover 18% of the chromosome 12 sequence. Bowman et al. were able to identify a region of extremely low (G plus C) content as the best candidate location for the centromere of P. falciparum chromosome 3. Our chromosome 12 sequence contains an analogous region between base positions 1,282,701 and 1,284,791 (2,090 bp; 0.092% of chromosome 12). That region has a (G plus C) content of 1.9%, is composed of the short tandem repeats characteristic of centromeres, and is, therefore, the putatative centromere of P. falciparum chromosome 12. To predict the genes encoded by P. falciparum chromosome 12, we used 'gene-calling' software in parallel with our colleagues at the WTSI2. Plasmodium falciparum chromosome 12 is predicted to encode 529 genes, including 23 genes from known Plasmodium-specific protein-encoding gene families (eight vars, twelve rifs, and three stevors) and three transfer RNA genes. The segmental (G plus C) content affects the speed and accuracy of sequencing. The predicted exons are, on average, 23.8% (G plus C), which is significantly higher than the overall average (19.3%). The predicted introns are, on average, 13.4% (G plus C), which is significantly lower than the overall average(Hall et al., 2002).
    2. Mitochondrion(Website 32)
      1. GenBank Accession Number: NC_002375
      2. Size: 5967 bp(Website 32).
      3. Gene Count: 3 genes(Website 32).
      4. Description: The complete 6 kb mitochondrial genome was sequenced from the single known isolate of P. reichenowi and from four different cultured isolates of P. falciparum, and aligned with the two previously derived P. falciparum sequences. The extremely low synonymous nucleotide polymorphism in P. falciparum (small pi, Greek equals 0.0004) contrasts with the divergence at such sites between the two species (K equals 0.1201), and supports a hypothesis that P. falciparum has recently emerged from a single ancestral population(Conway et al., 2000).
      5. Picture(s):
        • Plasmodium falciparum mitochondrion, complete genome (Website 32)



        • Plasmodium falciparum mitochondrion, complete genome (Website 32)



    3. Plastid(Website 61, Website 62)
      1. GenBank Accession Number: X95275 X95276
      2. Size: 15421 bp and 14001 bp(Website 61, Website 62). 34682 bp(Wilson et al., 1996).
      3. Gene Count: 67 genes(Williamson et al., 2002).
      4. Description: Malaria parasites and other members of the phylum apicomplexa harbour a relict plastid, homologous to the chloroplasts of plants and algae. The 'apicoplast' is essential for parasite survival, but its exact role is unclear. The apicoplast is known to function in the anabolic synthesis of fatty acids, isoprenoids and haeme, suggesting that one or more of these compounds could be exported from the apicoplast, as is known to occur in plant plastids. The apicoplast arose through a process of secondary endosymbiosis, in which the ancestor of all apicomplexan parasites engulfed a eukaryotic alga, and retained the algal plastid, itself the product of a prior endosymbiotic event. The 35-kb apicoplast genome encodes only 30 proteins, but as in mitochondria and chloroplasts, the apicoplast proteome is supplemented by proteins encoded in the nuclear genome and post-translationally targeted into the organelle by the use of a bipartite targeting signal, consisting of an amino-terminal secretory signal sequence, followed by a plastid transit peptide(Gardner et al., 2002).
    4. Plasmodium falciparum 3D7 chromosome 1(Website 3, Website 34)
      1. GenBank Accession Number: NC_004325
      2. Size: 643292(Website 34).
      3. Gene Count: 140(Website 3).
    5. Plasmodium falciparum 3D7 chromosome 2(Website 35, Website 36)
      1. GenBank Accession Number: NC_000910
      2. Size: 947102 bp(Website 36).
      3. Gene Count: 223(Website 35).
    6. Plasmodium falciparum 3D7 chromosome 3(Website 37, Website 38)
      1. GenBank Accession Number: NC_000521
      2. Size: 1060087 bp(Website 38).
      3. Gene Count: 237(Website 37).
    7. Plasmodium falciparum 3D7 chromosome 4(Website 39, Website 40)
      1. GenBank Accession Number: NC_004318
      2. Size: 1204112 bp(Website 40).
      3. Gene Count: 231(Website 39).
    8. Plasmodium falciparum 3D7 chromosome 5(Website 41, Website 42)
      1. GenBank Accession Number: NC_004326
      2. Size: 1343552 bp(Website 42).
      3. Gene Count: 312(Website 41).
    9. Plasmodium falciparum 3D7 chromosome 6(Website 43, Website 44)
      1. GenBank Accession Number: NC_004327
      2. Size: 1378756 bp(Website 44).
      3. Gene Count: 307(Website 43).
    10. Plasmodium falciparum 3D7 chromosome 7(Website 45, Website 46)
      1. GenBank Accession Number: NC_004328
      2. Size: 1351552 bp(Website 46).
      3. Gene Count: 262(Website 45).
    11. Plasmodium falciparum 3D7 chromosome 8(Website 47, Website 48)
      1. GenBank Accession Number: NC_004329
      2. Size: 1325595 bp(Website 48).
      3. Gene Count: 289(Website 47).
    12. Plasmodium falciparum 3D7 chromosome 9(Website 49, Website 50)
      1. GenBank Accession Number: NC_004330
      2. Size: 1541723 bp(Website 50).
      3. Gene Count: 355(Website 49).
    13. Plasmodium falciparum 3D7 chromosome 10(Website 51, Website 52)
      1. GenBank Accession Number: NC_004314
      2. Size: 1694445 bp(Website 52).
      3. Gene Count: 403(Website 51).
    14. Plasmodium falciparum 3D7 chromosome 11(Website 53, Website 54)
      1. GenBank Accession Number: NC_004315
      2. Size: 2035250 bp(Website 54).
      3. Gene Count: 486(Website 53).
    15. Plasmodium falciparum 3D7 chromosome 12(Website 55, Website 56)
      1. GenBank Accession Number: NC_004316
      2. Size: 2271477 bp(Website 56).
      3. Gene Count: 526(Website 55).
    16. Plasmodium falciparum 3D7 chromosome 13(Website 57, Website 58)
      1. GenBank Accession Number: NC_004331
      2. Size: 2732359 bp(Website 58).
      3. Gene Count: 669(Website 57).
    17. Plasmodium falciparum 3D7 chromosome 14(Website 59, Website 60)
      1. GenBank Accession Number: NC_004317
      2. Size: 3291006 bp(Website 60).
      3. Gene Count: 769(Website 59).
Biosafety Information
  1. Biosafety information for Plasmodium falciparum
    1. Level: Biosafety level 2(Website 118).
    2. Applicable: Laboratory-associated infections with Plasmodium spp. (including P. cynomologi); Trypanosoma spp.; and Leishmania spp. have been reported. Although no laboratory infections with Babesia spp. have been reported, they could result from accidental needlestick or from the bite of an infected tick.Although laboratory animal-associated infections are not common, mosquito-transmitted malaria infections do occur(Website 118).
    3. Precautions: Biosafety Level 2 practices and facilities are recommended for activities with infective stages. Infected arthropods should be maintained in facilities which reasonably preclude the exposure of personnel or their escape to the outside. Primary containment (e.g., biological safety cabinet) or personal protection (e.g., face shield) may be indicated when working with cultures of Leishmania spp., T. cruzi, or with tissue homogenates or blood containing hemoflagellates. Gloves are recommended for activities where there is the likelihood of direct skin contact with infective stages of the parasites listed(Website 118).
    4. Disposal: All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility(Website 119).
Culturing Information
  1. P. falciparum liver stage in human hepatoma cell line :
    1. Description: Liver parasites completed their development within a Thai human hepatoma cell line (HHS-102), and the presence of ring-form parasites in erythrocytes overlying the liver cell culture confirmed that an entire liver cycle was completed, culminating in the production of viable blood-stage parasites. The HHS-102 cell line allows investigation of the undefined liver stage of falciparum malaria previously unavailable in the laboratory(Karnasuta et al., 1995).
    2. Medium: The human hepatoma cell line (HHS-102) cells were seeded into a sex-well tissure culture palte at a concentration of 2.0 x 10(5) cells/well and were maintained in 3 ml/well of a complete culture medium (a 1:1 mixture of RPMI 1640 meduim with L-glutamine[Sigma, St. Louis, MO] and minimum essential medium with L-glutamine [Gibco, Grand Island, NY] supplemented with 2.2 mg/ml of sodium bicarbonate, 150 U/ml penicillin, 150 mg/ml of streptomycin, and 10% fetal bovine serum)(Karnasuta et al., 1995).
  2. Continuous culture :
    1. Description: Continuous culture of any species of malarial parasite was first obtained in 1976 with Plasmodium falciparum, most important of the four species causing human malaria. The methods then reported have been widely used and have undergone relatively little modification. They depend on the maintenance of human erythrocytes under conditions that support intracellular development of the parasites(Trager, 1994).
    2. Medium: The culture medium is RPMI-1640, originally developed for human white blood cells supplemented with HEPES buffer and with hypoxanthine(Trager, 1994).
    3. Optimal Temperature: Dishes or plates must be incubated at 37-38 C(Trager, 1994).
    4. Note: Serum is an essential and important part of the medium(Trager, 1994). The ABO type should of course be compatible with the cells being used. Type O cells can be used with any type of serum, and AB serum with any type of cells. AB serum is also useful if cultures are being initiated with infected Aotus monkey erythrocytes(Trager, 1994).
Epidemiology Information:
  1. Outbreak Locations:
    1. New data are presented from the Democratic Republic of the Congo, Tanzania, Ethiopia, and Zimbabwe on the increasing urban malaria problem and on epidemic malaria. Between 6% and 28% of the malaria burden may occur in cities, which comprise less than 2% of the African surface. Macroeconomic projections show that the costs are far greater than the costs of individual cases, with a substantial deleterious impact of malaria on schooling of patients, external investments into endemic countries, and tourism. Poor populations are at greatest risk; 58% of the cases occur in the poorest 20% of the world's population and these patients receive the worst care and have catastrophic economic consequences from their illness. The social vulnerability requires better understanding for improving deployment, access, quality, and use of effective interventions. Studies from Ghana and elsewhere indicate that for every patient with febrile illness assumed to be malaria seen in health facilities, 4 to 5 episodes occur in the community. Effective actions for malaria control mandate rational public policies; market forces, which often drive sales and use of drugs and other interventions, are unilkely to guarantee their use. Artemisinin-based combination therapy (ACT) for malaria is rapidly gaining acceptance as an effective approach for countering the spread and intensity of Plasmodium falciparum resistance to chloroquine, sulfadoxine/pyrimethamine, and other antimalarial drugs. Although costly, ACT ($1.20 to 2.50 per adult treatment) becomes more cost-effective as resistance to alternative drugs increases; early use of ACT may delay development of resistance to these drugs and prevent the medical toll associated with use of ineffective drugs(Breman et al., 2004).
    2. Malaria is a major public health problem in sub-Saharan Africa. The high-elevation areas in East Africa had infrequent epidemic malaria between the 1920s and the 1950s. Malaria epidemics were not reported between the 1960s and the early 1980s after a malaria eradication campaign. Although the lack of documented epidemics in the highlands between the 1960s and the early 1980s could have been due to the methods for health data recording at the health clinics, it is well recognized that a series of malaria epidemics have occurred in the East African highlands, including the highlands in western Kenya, Uganda, Ethiopia, Tanzania, Rwanda, and Madagascar. As a consequence of low immunity in the human population of the highlands, malaria epidemics have caused significant human mortality in both children and adults. Compared to the malaria situation between the 1920s and 1950s, the current pattern of malaria in the highlands is characterized by increased frequencies, expanded geographic areas, and increased case-fatality rates(Zhou et al., 2004).
    3. We report an epidemic of Plasmodium falciparum malaria in the remote valley of Bamian (altitude 2250 m-2400 m) in the central highlands of Afghanistan. A team of malaria experts from the World Health Organization and HealthNet International carried out the investigation. A total of 215 peripheral blood smears were obtained and 63 cases of malaria (90.5% P. falciparum, the remainder P. vivax) were confirmed. The study revealed that areas vulnerable to malaria in Afghanistan are more widespread than previously recognized. The area had been malaria-free until recently, when the disease appears to have been introduced as a consequence of protracted conflict and resultant population movement, and transmitted locally during the short summer months. The outbreak led to severe morbidity and high mortality in a province having only a few poorly-provisioned health care facilities(Abdur et al., 2003).
    4. In August 2000, a charter flight brought 224 refugees from Tanzanian refugee camps to Mirabel airport just north of Montreal, Quebec, Canada. These refugees were immediately dispersed to smaller communities throughout the province to hasten their integration into Quebec society. Within 3 to 4 weeks of their resettlement, the McGill University Centre for Tropical Diseases (TDC), which acts as a reference center for the province, noted a striking increase in requests for confirmatory testing and assistance in treatment. There appeared to be an outbreak of malaria across the province. Malaria parasites were identified in 15 blood smears (11 with Plasmodium falciparum, 2 with Plasmodium ovale, and 2 with mixed infection) from African refugees submitted to the TDC during first 6 weeks after their arrival. Parasitemias ranged from 0.3 to 7%. Six children had parasitemias of greater than 3%, and one pregnancy was complicated by a diagnosis of P. falciparum(Ndao et al., 2004).
    5. Based on the reports of 18 fever related deaths in Bhojpur Primary Health Centre (PHC) of Moradabad District (UP), a study was undertaken during September and October, 2000 to explore the possibility of outbreak of malaria in the area and the reasons of the outbreak. The slide positivity rate (SPR) and slide falciparum rate (SFR) in active fever surveys were found to be 84.7% and 82.2% respectively. Among children the average enlarged spleen and spleen rate were 1.9 and 27.4 per cent respectively. The mortality rate of Anopheles culicifacies in DDT, Malathion and Deltamethrin was 42.5, 86.7 and cent per cent respectively. The results of the study revealed that the outbreak was of malaria with predominance of Plasmodium falciparum causing several deaths in the affected villages(Shukla et al., 2002).
    6. Parasitologic and entomologic cross-sectional surveys were carried out during an outbreak of malaria between December 1998 and August 2000 in forest villages near the Mohkhed Primary Health Center in the Chhindwara District of Madhya Pradesh in central India. In December 1998, surveys showed that more than 70% of the fever cases had malaria, with 87% of the malaria caused by Plasmodium falciparum. The rate of enlarged spleens in children was 74.5%. In November 1999, 58% of the inhabitants were infected with malaria, with 80% of these cases caused by P. falciparum. Chloroquine resistance was seen in 23% of the cases. Anopheles culicifacies was the dominant mosquito species in all surveys (70-85%) and was resistant to DDT. The results indicate that the incidence of malaria in Chhindwara has increased gradually from 0.31 per 1,000 in 1990 to 6.75 per 1,000 in 2000(Singh et al., 2003).
    7. Haiti is the only Caribbean island where malaria, practically always due to Plasmodium falciparum, persists in an epidemic-endemic state(Raccurt, 2002).
    8. Malaria epidemics in highland areas of East Africa have resulted in significant morbidity and mortality in the past decade. Highland areas of East Africa have been characterized as having unstable transmission, with similar rates of infection and disease in children and adults(John et al., 2004).
  2. Transmission Information:
    1. From: Anopheles, Humans (at lifecycle stage: Sporozoite) , To: Homo sapiens, Humans (at lifecycle stage: Sporozoite) --(at lifecycle progression level: Sporozoite-Trophozoite)
      Mechanism: Malaria infection in humans is initiated with the bite of an infectious female mosquito, which inject sporozoites of Plasmodium species into the circulation. These sporozoites rapidly bind and invade liver cells and undergo rapid multiplication, leading to the release of thousands of infective merozoites(Raether et al., 1989).
    2. From: Homo sapiens, Humans (at lifecycle stage: Microgametocyte, Macrogametocyte) , To: Anopheles, Humans (at lifecycle stage: Microgametocyte, Macrogametocyte) --(at lifecycle progression level: Merozoite-Microgametocyte_Macrogametocyte)
      Mechanism: The journey of Plasmodium within the mosquito begins as the mosquito ingests gametocytes with the blood of an infected host(Ghosh et al., 2003).
    3. From: Homo sapiens, Humans , To: Homo sapiens, Humans
      Mechanism: Plasmodium sometimes is transmitted by means other than the bite of a mosquito. The blood cycle may be initiated by blood transfusion, by malaria therapy of certain paralytic disease, by syringe-passed infection among drug addicts, or, rarely, by congenital infection(Schmidt and Roberts, 1985). Transmission of malaria via blood transfusion is relatively uncommon in the U.S. Over the past 30 years the incidence has decreased and is now estimated at less than one case/million units. In most cases, the implicated donor had visited an endemic area or had emigrated from such an area. There were 1,402 cases of malaria reported in the U.S. in 2000. Two of those cases were congenital and two were blood related(Smith and Wright-Kanuth, 2003). In 1917, Wagner-Jauregg induced malaria for the first time to treat paresis. A soldier with tertian malaria (P. vivax) was admitted to the psychiatric clinic at the University Hospital for Nervous and Mental Disease in Vienna, Austria, in June 1917. Blood was passsaged to 3 patients with 'dementia paralytica', marking the beginning of malaria therapy. As a result of studies on the development of malaria therapy, Wagner-Jauregg was awarded the Nobel Prize for Medicine in 1927. The first detailed report of laboratory-based malarial studies associated with the treatment of neurosyphilitic patients in England was that of Colonel S. P. James (a member of the Malaria Committee of the League of Nations), and P.G. Shute in 1926. Because of objections to induction of malarial attacks by the direct inoculation of blood from patient to patient, they conducted extensive studies at the Horton Mental Hospital in Epsom on the use of bites of plasmodial-infected mosquitoes to induce malaria. The clinical, chemotherapeutic, and immunologic aspects of infections with P. vivax, P. falciparum, P. malariae, and P. ovale in some 2,500 patients observed in the Horton Hospital were reported(Collins and Jeffery, 1999). Eleven narcotic injectors from a prison in Saigon were hospitalized with falciparum malaria. Coma and intense parasitemia were common and eight patients died soon after admission. Two of three autopsied cases also had purulent pulmonary infections. No non-addicted prisoners were hospitalized for malaria. Nine more unsuspected falciparum infections were found among 29 other addicts in the prison. The clustering of malaria infections among narcotic injectors who had not been in malarious areas indicates that the malaria was transmitted by the common use of needles and syringes. Cerebral malaria in an addict may be misdiagnosed as drug intoxication. Malaria surveillance is recommended for the increasing addict population in the cities of Southeast Asia(Brown and Khoa, 1975). Congenital infection certainly occurs, but with very varying frequency in different parts of the world, and apparently in a way related to the absence or presence of immunity. In those places where malaria is endemic and relatively stable in character transmission to the child is very rare, and the many documented cases come mainly from places where the disease is less regular in occurrence, or less stable, or refer to mothers whose experience of the disease has been relatively recent(Russell et al., 1963).
  3. Environmental Reservoir:
    1. Humans:
      1. Description: The human reservoir of infection (during the non-transmission season) will depend on the rate of recovery from infection, which in turn depends on the extent of previous exposure and the immune reaction to infection. Both these factors will alter with age for a given intensity of transmission. Therefore, in the absence of novel infections, the human reservoir of infection will have an age-specific distribution(Paul et al., 2004).
      2. Survival: The prognosis in well-treated malaria is excellent, even under military conditions. For instance, the case mortality rate in American troops in the Second World War averaged less than 0.1 percent, on death per thousand hospital admissions for malaria. Untreated or poorly treated falciparum malaria in some areas may be fatal in up to 25 percent of cases(Russell et al., 1963). The prognosis of severe falciparum is poor, with a case fatality rate of 15-20% in African children. Mortality is higher when multiple syndromes of severe disease are present(Weatherall et al., 2002).
  4. Intentional Releases:
    1. Currently no intentional releases information is available.
Diagnostic Tests Information
  1. Organism Detection Test:
    1. Conventional light microscopy :
      1. Description: Conventional light microscopy is the established method for the laboratory confirmation of malaria. The careful examination by an expert microscopist of a well prepared and well stained blood film remains currently the 'gold standard' for detecting and identifying malaria parasites. In most settings, the procedure consists of: collecting a finger-prick blood sample; preparing a thick blood smear (in some settings a thin smear is also prepared); staining the smear (most frequently with Giemsa); and examining the smear though a micrroscope (preferably with a 100X oil-immersion objective) for the presence of malaria parasite(Website 120). The detection limit of microscopy is approximately 5-20 parasites per ul(Hanscheid and Grobusch, 2002). Thick smears are 20 to 40 times more sensitive than thin smears for screening of Plasmodium parasites, with a detection limit of 10 to 50 trophozoites/ul. Thin smears allow one to identify malaria species (including the diagnosis of mixed infections), quantify parasitemia, and assess for the presence of schizonts, gametocytes, and malarial pigment in neutrophils and monocytes(Trampuz et al., 2003). The establishment and maintenance of a reliable and efficient diagnostic facility for malaria at the primary health care level is dependent on many (or a combination of) factors, which can seriously degrade the utility and relevance of the malaria laboratory. These can be broadly summarized as follows: A. DEFICIENCIES IN PERSONNEL: inadequate training; poor supervision; bad management including the improper utilization of the available expertise and work time. B. SUBSTANDARD OR INAPPROPRIATE EQUIPMENT: incorrect or poorly coordinated specifications, particularly of microscopes; irregular maintenance and replacement of worn-out parts; poor quality control of stains and reagents; inappropriate supply schedules which either cause delays in the supply of material or accumulate stocks which exceed the shelf-life of the material. C. INAPPROPRIATE TECHNOLOGY: improper use of techniques (e.g. think films for routine malaria diagnosis) or the introduction of new technology which in use is less efficient at the peripheral level than well-established methods; continuation of programs and routines which were once relevant but do not meet the current needs of primary health care. D. ORGANIZATIONAL DEFICIENCIES: the maintenance of an efficient and relevant organizational structure calls for frequent review and analysis of the current aims and objectives of the malaria diagnostic facility as well as the necessary changes in staffing and methods to meet these requirements with the resources available. From time to time this may involve radically changing how personnel and other resources are utilized and overcoming the inertia to change at both local and higher levels(Payne, 1988).
  2. Immunoassay Test:
    1. Rapid Diagnostic Tests :
      1. Description: Various test kits are available to detect antigens derived from malaria parasites. Such immunologic ("immunochromatographic") tests most often use a dipstick or cassette format, and provide results in 2-10 minutes. These "Rapid Diagnostic Tests" (RDTs) offer a useful alternative to microscopy in situations where reliable microscopic diagnosis is not available. Malaria RDTs are currently used in some clinical settings and programs. However, before malaria RDTs can be widely adopted, several issues remain to be addressed, including improving their accuracy; lowering their cost; and ensuring their adequate performance under adverse field conditions. Malaria RDTs are currently not approved by the U. S. Food and Drug Administration (FDA) for use in the United States(Website 122). Dipstick antigen-capture assays are cost-effective for the management of P. falciparum malaria in specific conditions: in epidemics and emergencies; in mobile clinics; where laboratory services are inadequate; where first-line treatment is much more expensive than the dipstick assay; and in previously treated severe cases for whom blood films may have become negative. Unit cost will determine the future choice in many geographical areas(Guerin et al., 2002). A study was conducted to measure the overall performance of several rapid diagnostic tests for Plasmodium falciparum infection, in order to select the most appropriate test to be used in the field. A total of 742 patients attending the out-patient department of Mbarara Hospital with a clinical suspicion of malaria were included in the study. For each patient, a thick/thin film and 5 rapid tests based on the detection of histidine-rich protein II (HRP-II) (Paracheck Pf dipstick and device, ParaHIT f, Malaria Rapid and BIO P.F.) were performed. Outcomes were validity, inter-reader reliability and 'ease of use in the field', measured by both the general characteristics of the test and by the opinion of the readers. About half (57%) of the patients were positive for P. falciparum. The Paracheck Pf (dipstick and device) was considered as the most appropriate for the use in the field, being sensitive (97%), moderately specific (88%), reliable (kappa coefficient = 0.97), easy to use and cheap (about US $0.5/test). The ParaHIT f represented a good alternative(Guthmann et al., 2002).
    2. Indirect Fluorescent Antibody Test :
      1. Description: Malaria antibody detection is performed using the indirect fluorescent antibody (IFA) test. The IFA procedure can be used to determine if a patient has been infected with Plasmodium. Because of the time required for development of antibody and also the persistence of antibodies, serologic testing is not practical for routine diagnosis of acute malaria. However, antibody detection may be useful for: 1. Screening blood donors involved in cases of transfusion-induced malaria when the donor's parasitemia may be below the detectable level of blood film examination; 2. Testing a patient with a febrile illness who is suspected of having malaria and from whom repeated blood smears are negative; 3. Testing a patient who has been recently treated for malaria but in whom the diagnosis is questioned(Website 121).
  3. Nucleic Acid Detection Test: