Chapter 9 - Phylum Apicomplexa: Malaria

Taxonomy

P. Apicomplexa

C. Coccidia

O. Haemosporida

G. Plasmodium

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

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

•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

Anopheles sp.

Plasmodium sp. sporozoites

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

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

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

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

Erythrocyctic trophozoite

Merozoite entering erythrocyte

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

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

Erythrocytic Shizogonic Cycle cont.

One of 2 fates await these merozoites: 1. Become signet ring trophozoites and begin shizogony anew2. Differentiate into sexual stages, becoming male microgametocytes or female 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

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

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

• 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

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

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

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

Longitudinal section of mosquito intestine showing numerous oocysts

Sporozoites isolated from the salivary glands of a mosquito

Plasmodium vivax (benign tertian malaria)

• Less than 1% of the total RBC population is parasitized • Predilection for immature RBCs (reticulocytes)• Schuffner’s 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

P. ovale (mild tertian malaria)

•Less than 1% of the total RBC population is parasitized • Predilection for immature RBCs (reticulocytes)• Schuffner’s 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

Plasmodium malariae (quartan malaria)

• Parasitizes about 0.2% of older RBCs• Trophozoites accumulate pink staining Ziemann’s 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

• 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 Maurer’s dots (precipitates in the cytoplasm of RBCs infected to P. falciparum), tend to aggregate around the nuclear region of gametocytes

Plasmodium falciparum (Malignant tertian malaria)

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.

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

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

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); it’s 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

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 body’s 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

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

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

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 host’s 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 mother’s milk, they are at risk at the time of weaning• Also, P. falciparum can cross the placenta and cause infection on the fetus

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