Chapter 9 Phylum Apicomplexa: Malaria

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Chapter 9 - Phylum Apicomplexa Malaria

• Taxonomy
• P. Apicomplexa
• C. Coccidia
• O. Haemosporida
• G. Plasmodium

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• Overview
• Malaria is one of the most prevalent and
  debilitating diseases afflicting humans
• Worldwide prevalence is at approximately 489
  million cases, making malaria the most prevalent
  human parasitic disease, with an annual death
  toll of 2 million
• There are more than 50 species of Plasmodium,
  but only 4 commonly cause malaria in humans - P.
  vivax, P. falciparum, P. malariae, and P. ovale

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• Life Cycle Overview
• The life cycle of Plasmodium that infect humans
  includes 2 hosts
• 1) the human host and 2) the insect vector, a
  female mosquito belonging to the genus Anopheles

Anopheles sp.

• Like other apicomlexa, a significant feature of
  the life cycle is the alternation of sexual and
  asexual phases in the 2 hosts
• The asexual cycles, termed merogony, occur in
  the human
• The sexual cycle, termed gamogony occurs mainly
  in the mosquito
• Subsequent to the sexual stage, another asexual
  phase of reproduction occurs in the mosquito,
  termed sporogony

• The infective form in humans is the slender,
  elongated sporozoite

Plasmodium sp. sporozoites
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• Life Cycle (Detail)
• During feeding, the mosquito secretes
  sporozoite-bearing saliva beneath the epidermis
  of the human victim, thus inoculating the
  sporozoites into the blood stream

• About 24-48 hr later, sporozoites appear in the
  parenchymal cells in the liver, initiating the
  exoerythrocytic shizogonic cycle or
  pre-erthrythrocytic cycle

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• Exoerythrocytic Shizogonic Cycle
• Inside the liver cell, the sporozoite develops
  into a trophozoite, feeding on host cytoplasm
  with its functional micropore
• After 1-2 weeks, the nucleus of the trophozoite
  undergoes multiple fission, producing thousands
  of merozoites

• These rupture from the host cell, enter the
  blood circulation, and invade RBCs, initiating
  the erythrocytic shizogonic cycle
• Some sporozoites become dormant hypnozoites

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• Note
• Studies of P. vivax show that the membrane
  receptor site for the engulfment phenomenon is
  determined by the type of antigen present on the
  surface of the RBC - e.g., merozoite penetration
  requires the presence of at least one of two
  Duffy antigens (Fya or Fy b )
• People that lack the Duffy antigens (almost all
  West Africans and about 70 of American blacks)
  are resistent to vivax malaria
• However, P. ovale and P. falciparum malarias
  are not influenced by Duffy antigens, thus
  accounting for their prevalence in West Africa

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• Erythrocytic Shizogonic Cycle
• Electron microscopy has confirmed that
  merozoites interact with the RBC plasma membrane
  and actively invade the cell
• During this process, rhoptries and micronemes
  are believed to secrete surface active molecules
  that cause the host RBC membrane to expand and
  invaginate to form a parasitophorous vacuole
  which envelops the parasite

Merozoite entering erythrocyte
Erythrocyctic trophozoite
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• Erythrocytic Shizogonic Cycle cont.
• Once in the RBC, the merozoite assumes an early
  trophozoite shape consisting of a ring of
  cytoplasm and a dot-like nucleus - the signet
  ring stage

• These early trophozoites feed on host
  hemoglobin, grow to the mature trophozoite stage,
  and then undergo multiple fission as schizonts,
  producing a characterisitc number of merozoites
  in each infected RBC

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• Erythrocytic Shizogonic Cycle cont.
• Merozoites eventually rupture RBCs and each
  merozoite is capable of infecting a new RBC

Scanning electron micrograph of
Plasmodium-infected red blood cells
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Erythrocytic Shizogonic Cycle cont.
One of 2 fates await these merozoites 1. Become
signet ring trophozoites and begin shizogony
anew 2. Differentiate into sexual stages,
becoming male microgametocytes or female
macrogametocytes
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• Life Cycle cont.
• The sexual phase occurs in the female Anopheles
  mosquito and begins when the mosquito takes a
  blood meal that contains microgametocytes and
  macrogametocytes

• Once the surrounding RBC material is lysed, the
  gametocytes are released into the lumen of the
  stomach
• The microgametocytes undergo a maturation
  process known as exflagellation

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• Exflagellation
• The nucleus undergoes 3 mitotic divisions,
  producing 6-8 nuclei that migrate to the
  periphery of the gametocyte
• Accompanying the nuclear divisions are
  centriolar divisions, following which one portion
  joins each nuclear segment to become a basal
  body, providing the center from which the axoneme
  subsequently arises

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• Life Cycle cont.
• The nucleus with the axoneme and a small amount
  of cytoplasm form a microgamete, which detaches
  from the mass and swims to the macrogametocyte

• During this period the macrogametocytes have
  developed into macrogametes which become
  penetrated by the microgamete
• The fusion of male and female pronuclei
  (syngamy) produces a diploid zygote that
  elongates into a motile wormlike ookinete

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• Life Cycle cont.
• The ookinete penetrates the gut wall of the
  mosquito to the area between the epithelium and
  the basal lamina, where it develops into a
  rounded oocyst

• Growth of the oocyst is, in part, due to the
  proliferation of haploid cells called
  sporoblasts, within the oocyst

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• Life Cycle cont.
• Sporoblast nuclei undergo numerous divisions,
  producing thousands of sporozoites enclosed
  within the sporoblast membranes
• As membranes rupture, sporozoites enter the
  cavity of the oocyst

• The sporozoite-filled oocysts themselves
  rupture, releasing the sporozites in the hemocoel
• The sporozoites are carried to the salivary
  gland ducts of the insect and are ready to be
  injected into the next victim when another blood
  meal is taken

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Sporozoites isolated from the salivary glands of
a mosquito
Longitudinal section of mosquito intestine
showing numerous oocysts
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Plasmodium vivax (benign tertian malaria)

• Less than 1 of the total RBC population is
  parasitized
• Predilection for immature RBCs (reticulocytes)
• Schuffners dots usually stains pink to red when
  subjected to stains
• Hemozoin granules, by-products of hemoglobin
  degradation by the parasite, are prominent
• The cytoplasm of the trophozoite stages is very
  irregular and displays an active ameboid movement

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P. ovale (mild tertian malaria)

• Less than 1 of the total RBC population is
  parasitized
• Predilection for immature RBCs (reticulocytes)
• Schuffners dots usually stains pink to red when
  subjected to stains
• Hemozoin granules, by-products of hemoglobin
  degradation by the parasite, are prominent
• The cytoplasm of the trophozoite stages is very
  irregular and displays an active ameboid movement

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Plasmodium malariae (quartan malaria)

• Parasitizes about 0.2 of older RBCs
• Trophozoites accumulate pink staining Ziemanns
  dots
• Hemozoin granules appear in the center or
  periphery of the shizont
• Trophozoite often appear as a band across the
  cell
• Mature trophozoites resemble macrogametocytes
• Recrudescensces as long as 52 years after
  initial infection

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Plasmodium falciparum (Malignant tertian malaria)

• Only ring trophozoites and gametocytes seen in
  peripheral circulation later stages trapped in
  capillaries of muscle and visceral organs
• Plasma membranes of infected RBCs undergo
  alteration causing them to adhere to the walls
  of capillaries
• Infects RBCs of any age about 10 of the total
  RBCs
• Multiple infections of single RBCs are common
• Gametocytes are crescent shaped cells
• Hemozoin as well as Maurers dots (precipitates
  in the cytoplasm of RBCs infected to P.
  falciparum), tend to aggregate around the nuclear
  region of gametocytes

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• Epidemiology
• Endemicity of human malaria is usually
  determined by the geographic distribution of its
  anophelene mosquito areas where the vector is
  not present are free of the disease
• Local environmental factors determine which
  particular species of mosquito transmits malaria
  in a given area local epidemiological surveys
  can be used to assay the prevalent vectors
• Precipitin tests of ingested blood from infected
  mosquitoes reveal whether the vectors have
  zoophilic or anthrophilic feeding preferences
• Water dependency for breeding varies greatly
• The control of malaria depends on a variety of
  factors, such as availability of antimalarial
  drugs, use of screens on houses to keep out
  mosquitoes, proper use of insecticides,
  elimination of mosquito breeding sites, etc.

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• Relapse of Infection
• Victims of vivax or ovale malaria may suffer a
  relapse
• Originally, the relapse was thought only to be
  due to populations of cryptozoites
  (pre-erythrocyte shizont) being maintained in the
  exoerythrocytic cycle
• While one population progressed to the usual
  erythrocytic phase, underwent shizogony and
  released merozoites into the circulating blood
  stream causing malaria, the other population
  maintained an ongoing exoerythocytic cycle known
  as a para-erythrocytic cycle
• Parasites in the hepatic stages of the cycle
  were thought to be protected from the host
  antibodies until activated by some physiological
  change within the host that allowed them to erupt
  from the hepatocytes, precipitating another bout
  of malaria
• A more recent view also recognizes the existence
  of 2 different populations of sporozoites
• Short prepatent sporozoites - upon entering the
  human host, undergo the usual exoerythrocytic and
  erythrocytic phases of development and cause
  malaria
• Long prepatent sporozoites or hypnozoites -
  remain dormant in the hepatocytes for an
  indefinite period
• Some kind of physiological fluctuation activates
  them into exoerythrocytic and erythrocytic cycles
  and a relapse occurs

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• Recrudesence
• Recurrence of malaria among victims infected by
  P. malariae many years after apparent cure
  fostered the idea that this species produced
  relapses like those produced by P. vivax and P.
  ovale
• But, it has been shown that the periodic
  increase in numbers of parasites results from a
  residual population persisting at very low levels
  in the blood after inadequate or incomplete
  treatment of the initial infection
• The situation may persist for as long as 53
  years before something triggers a parasite
  population explosion with accompanying disease
  manifestations -
• This phenomenon is referred to as recrudesence

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• Symptomatology and Diagnosis
• Pathology in human malaria (P. falciparum) is
  generally manifested in 2 basic forms host
  inflammatory reactions and anemia
• Host inflammatory reactions are initiated by the
  periodic rupture of infected RBCs, which release
  malarial pigment such as hemozoin and parasite
  metabolic wastes
• These ruptures are accompanied by fever
  paroxysms that are usually synchronous except
  during the primary attack (correlated with the
  merozoites rupturing from RBCs)
• During cell rupturing, toxins are released which
  in turn cause macrophage cells to release tumor
  necrosis factor (TNF) its TNF that actually
  induces the fever
• During the primary attack synchrony may not be
  evident, since the infection may arise from
  several populations of liver merozoites at
  different stages of development

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• Symptomatology and Diagnosis cont.
• Macrophages, particularly those in the liver,
  bone marrow, and spleen, phagocytose released
  pigment
• In extreme cases the amount of pigment is so
  great that it imparts a dark green, reddish brown
  hue to the visceral organs such as the liver,
  spleen and brain
• With increased RBC destruction, accompanied by
  the bodys inability to recycle iron bound in the
  insoluable hemozoin, anemia develops
• TNF toxicity may also induce splenic removal of
  unparasitized RBCs and inhibit bone marrow
  production of new RBCs
• One pathological element unique to P. falciparum
  is vascular obstruction
• Plasma membranes of RBCs infected with schizonts
  develop electron dense knobs by which they
  adhere to the endothelium of capillaries in
  visceral organs

• As a consequence, the capillaries become
  obstructed, causing the affected organs to become
  anoxic
• In terminal cases, blocked capillaries in the
  brain (cerebral malaria) cause it to become
  swollen and congested

Infected RBC showing surface knobs
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• Black Water Fever
• A condition known as black water fever often
  accompanies falciprum malaria infections
• It is characterized by massive lysis of RBCs
  and it produces abnormally high levels of
  hemoglobin in urine and blood
• Fever, vomiting with blood, and jaundice also
  occur
• There is between 20-50 mortality rate, usually
  due to renal failure probably due to renal
  anoxia
• The exact cause of this condition is uncertain
• It may be a reaction to quinine, or it may
  result from an autoimmune phenomenon in which
  hemolytic antibodies are produced in response to
  chemotherapy

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• Chemotherapy
• Malaria control requires effective treatment of
  the disease in humans and continuous efforts to
  control mosquito populations
• The first known antimalarial drug was quinine
• The drug primarily destroys the schizogonic
  stages of malaria
• The synthetic drug Atabrine dihydrochloride
  (circa 1936-36) proved useful against
  erythrocytic stages and in suppressing clinical
  stages
• Since WWII several synthetic drugs have been
  used chloroquine, amodiaquin, and primaquine
• Chloroquine is a weak base and it increases the
  pH of the food vacuole which in turn prevents the
  digestion of hemoglobin
• Pyrimethamine used in combination with
  sulfadoxine have been effective in inhibiting the
  folic acid cycle of malarial parasites

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• Immunity
• In addition to chemotherapy research,
  development of a protective vaccine against
  malaria is being pursued
• Interestingly, the surface coat of the
  sporozoite acts as a renewable decoy to the
  vertebrate hosts immune system, stimulating the
  production of antibodies
• When the sporozoite is attacked and its decoy
  coat sloughs off, a replacement coat is
  synthesized and its decoy effect continues
• This system provides ideal protection for the
  sporozoite which only spends a brief amount of
  time in the blood before it penetrates a liver
  cell as is protected from circulating antibodies
• In endemic areas, premunition is the basis for
  protective immunity as long as low-level
  infection persists however, with complete cure,
  the victim regains susceptibility
• Also, while nursing infants in endemic areas are
  protected through antibodies in their mothers
  milk, they are at risk at the time of weaning
• Also, P. falciparum can cross the placenta and
  cause infection on the fetus

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• Genetics and Malaria Infections
• Several genetic conditions are known to affect
  the malarial organism
• Susceptibility is conferred by the presence of
  Duffy antigens e.g., vivax merozoite
  penetration of RBCs requires 1 of 2 Duffy
  antigens
• Genetic deficiency in G6PDH activity in RBCs
  (favism) creates and inhospitable environment for
  the parasites
• Humans heterozygous for sickle cell anemia
  possess a selective advantage over individuals
  with normal hemoglobin in regions where P.
  falciparum is endemic