Wednesday, October 3, 2012

Introduction to Protozoa

Introduction to Protozoa

Protozoa are unicellular eukaryotes that seem to live as parasites in all species of multicellular organisms, and some even parasitise other unicellular organisms. Some are intracellular parasites, while others live in extracellular locations such as body fluids, blood or lumen of the host animal. They phylum Protozoa is divided into four classes, of which three are important.
1. Class Mastigophora – consists of flagellates that are equipped with whip-like locomotor structures. The arrangement and number of these flagella are used in classification. Some of the flagellates are classified as plants. Indeed subclass Phytomastigina contains plant like flagellates, which have pigmented bodies called chromoplasts, or chromatophores in which photosynthesis occurs. Like other flagellates, the Phytomastigina move by one or more flagella. Some of them form colonies, thus suggesting an evolutionary trend towards multicellularity.
Subclass Zoomastigina contains animal-like flagellates. They lack chromatophores and are divided into several orders:
Order Rhizomastigida consists of flagellates that move by amoeboid movement, using pseudopodia.
Order Protomastigida is made up of simple flagellates. Included in this group is the family Trypanosomidae that includes disease-causing genera Trypanosoma, Schizotrypanum and Leishmania.
Order Polymastigida contains uninucleate and binucleate species as well as some with polynuclei. There may be as many as five to eight flagella; all must have at least three. This Order includes the family Hexamitidae, to which the intestinal flagellate Giardia, belongs
Order Trichomonadida consists of either uninucleate or multinucleate flagellates with internal rod-like axostyle and a flagella complex of 3 to 6 flagella and a prominent parabasal body. The group contains a number of parasites of man and animals, including Trichomonas vaginalis.
Order Hypermastigida consists of uninucleate flagellates with many flagella. These are symbiotic parasites of wood eating termites
Order Opalinida includes parasites of amphibians.
2. Class Sarcodina- includes free-living and parasitic amoebae of the order Amoebida. Family Endomoebidae contains the genus Amoeba that is parasitic in man. This includes Entamoeba histolyticaE. coli and E. gingivalis.
3. Class Ciliata contains ciliated organisms. The cilia are used for locomotion. Ciliates possess two kinds of nuclei - the macronucleus and the micronucleus. The macronucleus interacts with the cytoplasm in the control of metabolism and growth, while the micronucleus functions in reproduction .The only ciliate parasitic to man is Balantidium coli.
4. Class Sporozoa consists of spore-forming protozoa that are enclosed in tough membranes that are quite resistant to adverse conditions, and can be dispersed by wind and water. They also produce sporozoites that can only survive in the host. Included in this group are malaria parasites and coccidia.

Malaria( Plasmodium vivax,P. falciparum, P. malariae and P. ovale)


Malaria scourge

Malaria is one of the most important parasitic diseases of man. It has a wide distribution in AfricaAsia, the Middle East, Central and South America, the Caribbean and the Pacific Islands. The malaria agent, Plasmodium, is a protozoan parasite that is transmitted by female Anopheles mosquitoes. Four species of Plasmodium are parasites of man, namely Plasmodium vivax,P. falciparumP. malariae and P. ovaleP. vivax accounts for about 40% of the world’s malaria cases and 5 – 10 % of the malaria cases in East Africa.
Plasmodium falciparum is the most virulent of the malaria parasites and is often fatal. It contributes up to 50% of all the malaria cases in the world and more than 90% of malaria cases encountered in East Africa. Both P. malariae and P. ovale have a low prevalence, accounting for less than 10% of the world’s malaria cases.
The number of people at risk of contracting malaria in the world is estimated at two billion. Of these 270 million are infected while the number of those showing clinical symptoms is around 110 million. The annual mortality is around 1-2 million, mainly among children under five years of age. The majority of these children are found in the sub-Saharan Africa.
The socioeconomic aspects of malaria are significant. In the endemic areas, morbidity is high, rendering a large section of the population economically unproductive. Constant malaria attacks sap the energies of the population and adversely affect its economic productivity Mortality among the vulnerable groups can be frighteningly high.
Money, which is usually scarce, is spent on treatment and on provisions needed to nurse the sick at the expense of other pressing needs. If the breadwinner is sick, the family goes hungry. Absenteeism due to sickness becomes a problem and the children so affected perform badly in school.
Life cycle
Female Anopheles mosquitoes transmit malaria. There are many species of Anopheles involved depending on the region.Anopheles gambiae complex and Anopheles funestus are the main transmitters in AfricaA. gambiae is a particularly an effective transmitter of malaria. It has a wide range of breeding sites such as temporary ponds, potholes, plant leaves, discarded motor tyres, and cans. Under warm, wet conditions, the entire life cycle can take less than two weeks.
Malaria life cycle involves female Anopheles mosquito and man
Mosquitoes become infected when they suck blood from people who carry gametocytes. The gametocytes are the sex forms of the malaria parasites. When the mosquito ingests blood that contains gametocytes, the latter mature in the mosquito’s midgut into microgametes and macrogametes. The microgamete behaves as a sperm cell, the macrogamete as an ovum. The microgametes are flagellated and are formed by a process known as exflagellation. Exflagellation involves the male leaving the host erythrocyte and extruding about eight microgametes, each resembling a long flagellum. After a few minutes, each of these flagellated microgametes swims about and seeks a female gamete. The process of exflagellation is usually completed within ten to fifteen minutes and is immediately followed by fertilisation.
The zygote formed is active and is known as ookinete. The ookinete, a somewhat elongated and worm-like organism penetrates the insect’s gut wall and grows by a form of nuclear division known as schizogony. About three to four weeks later, a sporocyst, which is a kind of a sac that contains thousands of sporozoites, is formed. Later the sporocyst ruptures and releases spindle shaped sporozoites. Released sporozoites migrate to the salivary glands where they will be transferred to a host when the mosquito takes another blood meal.
Exoerythrocytic stage
when an infected mosquito bites, it introduces its saliva together with the sporozoites into the victim’s blood. the saliva contains a coagulant that prevents the blood from clotting so that it does not block the fine proboscis tube. after inoculation, the sporozoites circulate in the blood stream for period of 30 minutes before disappearing within the liver parenchymal cells. once inside the liver, the parasites undergo division and change into schizonts. the schizonts absorb nutrients from the liver cells, become greatly enlarged, and 7 to 8 days after infection, thousands of merozoites appear. this hepatic phase is known as the exoerythrocytic stage.
                                          A stained blood smear shows ,among others, ring forms (1) and gametocytes (2)
Erythrocytic stage
The merozoites spill over into the blood circulatory system, invade the red blood cells and transform into trophozoites. The erythrocytic stage starts with the attachment and penetration of the red blood cells by the merozoites.
each trophozoite is a minute blob of chromatin and a little cytoplasm, which assumes a ring form. stained with giemsa the chromatin appears red and the cytoplasm blue. the young ringlets grow and, in the case of p. vivax, increasingly exhibit amoeboid activity. eventually the parasite rounds up and forms a schizont.
The erythrocytic merozoites feed by ingesting haemoglobin, accumulating malaria pigment as they grow. The chromatin divides by schizogony or segmentation until the chromatin mass is too large for the cell, which ruptures releasing thousands of merozoites into the blood stream. Each erythrocytic cycle in P. vivaxP. falciparum, and P. ovale takes 36 – 48 hrs while that of P. malariae is synchronised at 72 hrs. Some of the merozoites that enter the red blood cells develop into gametocytes. Gametocytes develop only in the vertebrate host.
All the merozoites of P. falciparum leave the liver and enter the blood circulation unlike those of the other species, particularlyP. vivax and P. ovale that leave some parasites in the liver cells in a dormant state for months, or even years and give rise to relapsing malaria attacks. .
Because of the risk of relapsing malaria, some countries do not allow travellers to malarious areas to donate blood for up to three years after returning home.
Pathogenesis
The pathogenicity of malaria is related to the erythrocytic infection. The plasmodia in red blood cells grow and segment at the expense of the host’s cells. As the number of parasites increases, with each successive schizogony, the number of erythrocytes decreases due to rupture of the parasitised cells and lysis of nonparasitised cells. As the number of invaded red blood cells increases and the asexual cycles of the parasites become more synchronised, characteristic chills and fever associated with malaria attack appear. The speed with which symptoms develop depends on the species of Plasmodium and on the host’s immune reaction.
P. falciparum is the cause of most of the fatalities associated with malaria. It invades erythrocytes of all ages and is thus capable of producing very high parasiteamias. It has a schizogonic cycle that is not more than 48 hours but frequently less synchronised. Moreover, in P. falciparum infections there is a tendency for more than one parasite, frequently several, to develop in a single red blood cell. Thus in two or more asexual cycles, the number of infected red cells frequently reaches a dangerous threshold, often before the usual symptoms such as fever and chills become apparent.
In addition, erythrocytes containing P. falciparum parasites tend to adhere to one another and to the lining of blood vessels, causing blockage of blood capillaries in vital areas such as the brain, lungs and kidneys, while toxic products interfere with oxygen utilisation by the host cells.
Destruction of the red corpuscles by the parasites leads to anaemia. In pernicious or serious malaria, destruction and agglutination of the parasitised red blood cells may reduce the number of the erythrocytes to a fifth of the normal. This progressive decrease in the number and quantity of circulating erythrocytes, with corresponding reduction in oxygen transport, causing oxygen starvation of the tissues, is followed by multiple thromboses in the smaller blood vessels and progressive decrease in circulating blood volume.
The placenta has a heavy concentration of plasmodia in all stages of development in the maternal blood sinuses. The spleen and liver are characteristically enlarged in malaria infections.
Symptoms
The symptoms of malaria start as flu usually 8 to 30 days after being inoculated with parasites by an infected mosquito. They are characterised by cycles of fever, shaking, chills and sweating. The symptoms may subside. The frequency of the symptoms depends on the species of malaria parasite involved. In P. vivaxP. ovale and P. falciparum, they appear every 2 days, whereas in P. malariae, they occur every 3 days.
The periodicity of the symptoms coincides with the destruction of the red blood cells and the release of haemoglobin and other substances, some of which are probably poisonous. The pathogenesis of malaria is therefore related to the destruction of the red blood cells and the effect of the debris of the ruptured cells and metabolic products released into the circulation. P. vivax and P. ovale tend to invade young red blood cells while P. malariae seems to prefer older red blood cells. P. falciparum, on the other hand, infects red blood cells of all ages. As a result, this species produces the most severe malaria and accounts for a large proportion of the fatalities associated with malaria.
The incubation period is usually 8 to 15 days. Symptoms due to P. falciparum are associated with chills, shivering, high fever and profuse sweating. Other symptoms include headache, bone and muscular pains, malaise, anxiety, mental confusion and even delirium. Leucopoenia and severe anaemia are common. Blood examination shows the predominance of ring stages and gametocytes.
As the infection progresses, the symptoms intensify. The hyperpyrexia or high fever may appear, characterised by hot, dry, pale skin covered with sticky sweat. The skin feels cold but the temperature may be as high as 39 C. Other signs include vomiting diarrhoea, low blood pressure and oedema.
Cerebral Malaria
Cerebral malaria is an acute disease of the brain, which is accompanied by fever, convulsions and paralysis or coma. It is due to the sequestration of brain capillaries and venues with parasitised red blood cells and nonparasitised red blood cells.
Cerebral malaria is more frequent in children under nine years of age and visitors as well as immigrants from nonmalarious areas. Therefore, any cases of mental symptoms in endemic areas should arouse suspicion of malaria first until it has been excluded.
It is only associated with infections that are caused by P. falciparum which appear to cause the membranes of infected red blood cells to become sticky, causing them to stick to and obstruct the small blood vessels of the brain. Ring-like lesions in the brain are a common characteristic of cerebral malaria.
Sickle Cell Anaemia
Common inherited blood disorder is sickle cell anaemia that is caused by the presence of abnormal haemoglobin in the red blood cells. The sickle cell trait is due to a defective gene on the haemoglobin molecule that is inherited as a recessive gene. People who inherit both recessive genes from their parents suffer from sickle cell anaemia but those who inherit one normal gene from one parent and one abnormal gene from the other parent are free from sickle cell anaemia. They are, however, carriers of the sickle cell trait. Those people who inherit only a normal gene from each of their parents are completely free from sickle cell anaemia.
The carriers of the sickle cell trait are protected from attacks of P. falciparum and this has been recognised for a long time among the African Americans and immigrants from the Middle East to North America who must have carried the protective gene with them from their malaria endemic native lands.
European immigrants to America and American Indians are not protected against falciparum malaria. The reason for this is not clear but the sickle cell may be deficient in nutritional factors needed to nourish the malaria parasite, in addition to being a poor oxygen carrier.
Endemicity
Endemicity of malaria refers to the degree of prevalence, as regards frequency and intensity, of malaria infection in the natural population in an area. However, if malaria is endemic in an area it means it is always present in that area, perhaps in slowly varying degrees of incidence and intensity , but often to a degree that is measurable in terms of morbidity or splenomegaly.
An epidemic of malaria is caused by a rapid increase in morbidity and mortality in the population. Epidemics occur when there have been increases in incidence and prevalence of cases of malaria, which may have been caused by climatic changes, disasters such as floods, or other human factors.
In endemic areas where malaria rates remain high and relatively stable throughout the year, most people have developed immunity and many have become asymptomatic carriers. This explains why in endemic areas the number of infected people is always greater than the number of people showing clinical symptoms. This may indicate that the population has developed immunity that suppresses parasite multiplication in the blood so that the infection is not heavy enough to generate symptoms of malaria.
However, vulnerable groups like the young children still carry high parasiteamias and suffer from severe attacks of malaria frequently. These groups need to be given particular attention, just as the same attention should be extended to pregnant mothers, visitors and migrant workers who come from nonmalarious areas.
Highland areas sometimes experience a typical form of malaria that is unstable and characterized by epidemic conditions. Under normal climatic conditions, the temperatures are usually not ideal for mosquito breeding. However, occasionally the seasons may become quite warm and wet, resulting in a substantial increase in mosquito numbers within a relatively short time. This is followed by an equally abrupt outbreak of malaria of epidemic proportions. Highland malaria is highly dangerous because it affects people who have very little immunity or none at all and can lead to many deaths before it is checked.
Diagnosis
Routine diagnosis of malaria involves taking thick and thin blood smears, and staining them with Giemsa stain. The stained smears show the presence of different forms of malaria parasites, such as merozoites (ring forms), schizonts and gametocytes. The erythrocytic forms of the various species of the malaria parasite have certain characteristics that distinguish them from one another.
P. Falciparum is characterised by ring-shaped stages (signet ring), sickle-shaped gametocytes and Maurer’s dots.
P. vivax has young ring-shaped stages, multinucleate amoeboid dividing stages with finely granular pigment. These are not present in P. falciparum. They also have the morula stage from which merozoites will arise. Infected erythrocytes show red, orange Schuffner’s stripling. P. malariae has ring form young stages that contain coarse granular yellow pigment. The multinucleated schizonts are barrel-shaped. The gametocytes are similar to those of P. vivax.
Treatment
Drugs used to treat malaria include chloroquine, quinine, fansidar (salfodoxine-pyrimethamine), mefloquine, halofantrine and artemisinin. The problem with these drugs is that all of them are threatened by the development of resistance. Although chloroquine has been the drug of choice against P. falciparum for a long time, its use is now very much hampered by resistance.
Artemisinin extracted from the Chinese plant, Artemisia annua, and melfoquine are currently used to treat multidrug resistant malaria. Proguanil, pyrimethamine and chloroguanil are usually used for prophylaxis. Unfortunately, they have a relatively long half-life and parasites easily develop resistance to these drugs. However, they show a synergistic potency when used in combination with other antimalarial drugs. For example, the antimalarial drug fansidar is made by combining two other drugs, pyrimethamine and salfodoxine.
Drug Resistance
One importance aspect of malaria management is the ease with which the malaria parasites have become resistant to the antimalarial drugs. In much of the Southeast Asia, the strains of P. falciparum have become resistant to almost all the antimalarials in use. This makes the management of malaria a tricky one, as a relentless search for new antimalarials must be kept up. The development of resistance in malaria is due to mutation and the use of subdoses to which the parasites become adapted.
Vector Control
(a) Larval control
Vector control aims at limiting the numbers of larvae and adult mosquitoes. Mosquitoes breed wherever there is standing water, such as potholes, ponds, obstructed drainage channels, discarded vehicle tyres, bushes and leaves. Therefore, the strategy should be to deny mosquitoes breeding sites. This can be done by filling up potholes with soil, getting rid of empty cans, drums, motor tyres and any water receptacles that can serve as breeding sites. Pouring oil such as used diesel on stagnant water destroys mosquito larvae. Water and sewerage drainage systems should be maintained regularly to allow unhampered flow and thus prevent mosquito breeding in them.
(b) Adult mosquito control
Although the mosquito bites habits are diverse, the majority of the mosquitoes bite indoors at night and rest on the walls and ceilings of houses after feeding. Therefore, by spraying the inner walls of houses with an insecticide with a lasting residual effect, the frequency of mosquito bites can be reduced substantially which inevitably causes a reduction in malaria transmission.
In addition, body creams containing insecticide as well as bednets impregnated with insecticides such as permethrin have been found quite effective in preventing mosquito bites as well reducing the rate of transmission and malaria attacks. Insecticide fumigants are also frequently used in homes.
The market is full of insecticides that can kill mosquitoes but their extensive use is very much constrained by environmental considerations.
Biological control has been used to control malaria with some degree of success. Fish such as guppies that breed rapidly and eat mosquito larvae are usually introduced in water tanks and other enclosed water bodies. Water-borne bacteria such asBacillus thuringiensis and Bacillus sphaericus are used as biological control agents because they produce toxins that kill mosquito larvae. Other biological control agents that have been attempted include viruses, protozoa, fungi and nematodes