MALARIA

INTRODUCTION
Malaria is a mosquito-borne infectious disease of humans and animals.

Origin of the word MALARIA:

The term malaria originates from Medieval Italian word. It means bad air. It was first used in English by H.Walpole when describing the disease and was used as MAL ARIA. The term was shortened to MALARIA in the 20th century. C.Laveran in 1880 was first to identify the parasites in human blood. In 1889, R. Ross discovered that mosquitoes transmitted malaria.

Malarial parasites:
Malaria is caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within the red blood cells causing symptoms that typically include fever and headache and in severe cases progressing to coma and death.

Plasmodium:
Five species of Plasmodium can infect & be transmitted by humans. They are:

 Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malariae Plasmodium knowlesi

Plasmodium falciparum;
It is a protozoan parasite and transmitted by the female Anopheles mosquito. Malaria caused by this species is also called Malignant or falciparum malaria & it is the most dangerous form of malaria with the highest rates of complications and mortality. Plasmodium falciparum causes severe malaria via a distinctive property not shared by any other human malaria, that of sequestration. Within the 48 hours asexual blood stage cycle, the mature forms change the surface properties of infected red blood cells, causing them to stick to the walls of blood vessel (a process called cytoadherence). This leads to obstruction of the microcirculation and results in dysfunction of multiple organs, typically the brain in cerebral malaria. Known vectors are Anopheles gambiae, Anopheles albimanus, Anopheles freeborni, Anopheles maculatus and Anopheles stephensi.

Plasmodium vivax:
Plasmodium vivax is a protozoan parasite and a human pathogen. The most frequent and widely distributed cause of recurring (tertian) malaria, P. vivaxis one of the four species of malarial parasite that commonly infect humans. It is less virulent than Plasmodium falciparum, which is the deadliest of the four, and is seldom fatal. P. vivax is carried by the female Anopheles mosquito, since it is the only sex of the species that bites.

Plasmodium ovale;
Plasmodium ovale is a species of parasitic protozoa that causes tertian malaria in humans. It is closely related to Plasmodium falciparum and Plasmodium vivax, which are responsible for most malaria. It is rare compared to these two parasites, and substantially less dangerous than P. falciparum. P. ovale has recently been shown by genetic methods to consist of two subspecies, P. ovale curtisi and P. ovale wallikeri.

Plasmodium malariae;
Plasmodium malariae is a parasitic protozoa that causes malaria in humans. It is closely related to Plasmodium falciparum and Plasmodium vivax which are responsible for most malarial infection. While found worldwide, it is a so-called

"benign malaria" and is not nearly as dangerous as that produced by P. falciparum or P. vivax. P. malariae causes fevers that recur at approximately three-day intervals (a quartan fever); longer than the two-day (tertian) intervals of the other malarial parasites, hence its alternate names quartan fever and quartan malaria.
Plasmodium knowlesi;

Plasmodium knowlesi is the fifth major human malaria parasite. It may cause severe malaria as indicated by its asexual erythrocytic cycle of about 24 hours; with an associated fever that typically occurs at the same frequency (i.e. the fever is quotidian). This is an emerging infection that was reported for the first time in humans in 1965. It accounts for up to 70% of malaria cases in certain areas in South East Asia where it is mostly found. This parasite is transmitted by the bite of an Anopheles mosquito. Plasmodium knowlesi has health, social and economic consequences for the regions affected by it. Plasmodium knowlesi is also a dangerous species that is typically found only in long-tailed and pigtail macaque monkeys.

Life cycle of Plasmodium:

Only female pregnant mosquitoes feed on blood seeking nutrients from blood to nourish her developing eggs while male

mosquitoes feed on plant nectar, thus males do not transmit the disease. The females of the Anopheles genus of mosquito prefer to feed at night. They usually start searching for a meal at dusk, and will continue throughout the night until taking a meal. If she drinks blood from someone infected with malaria, she too becomes infected with the disease. The life cycle of malaria is completed in the body of two hosts: i). Man: They are the secondary hosts ii). Female Anopheles mosquito: they are primary and definitive hosts and also serve as transmission vectors.
Life cycle of Plasmodium in Man:

When a female Anopheles mosquito bites a healthy person if transfers its sporozoites into the blood of man. The sporozoites are very small spindle, sickle-shaped bodies. They contain cytoplasm and nucleus.

Stages of life cycle in man;

In man, asexual life cycle of Plasmodium takes place. This is called as Shizogony. Schizogony comprises of following phases:

 Pre-erythrocytic phase:
Once within the human blood, the sporozoites invade the hepatic cells within an hour. After penetrating the hepatic cells, each sporozoite grows for number of days and becomes a shizont. It divides to form a large number of uninucleate exoerythrocytic merozoites. Within 6 to 12 days, the liver cells bursts and these merozoites are liberated which then invade fresh liver cells and multiply producing enormous number of new generation.

Erythrocytic phase:
It takes place in RBCs. The exoerythrocytic merozoites after escaping into the blood stream, invade the red blood corpuscles. Each becomes rounded and is called trophozoite. When it grows in size, the nucleus is pushed to one side into the peripheral cytoplasm. It resembles a signet ring and is referred to as signet ring stage. The trophozoite ingests a large amount of cytoplasm of the RBCs. The blood hemoglobin is broken down into its protein component, which is used by the trophozoite. It develops into an active amoeboid trophozoite. After active feeding it becomes rounded, grows in size and becomes a shizont. It now undergoes shizogony and produces erythrocytic merozoites. With the rupture of RBC, the erythrocytic merozoites along with malarial pigments

formed by the residue of hemoglobin are liberated into the blood plasma. These erythrocytic merozoites invade fresh corpuscles to repeat the cycle. The time taken to complete the erothrocytic phase of life cycle depends upon the species of Plasmodium.

Gametocytogenesis:
After few erythrocytic generations, erythrocytic merozoites which invade the RBCs increase in size to become gametocytes; male or microgametocytes and female or macro gametocytes. The gametocyte does not divide, but remain within their host blood corpuscles until they are invaded by the vendor, in which they continue their development.

INCUBATION PERIOD
The time from the initial malaria infection until symptoms appear (incubation period) generally ranges from: 9 to 14 days for Plasmodium (P.) falciparum. 12 to 18 days for P. vivax and P. ovale.

 18 to 40 days for P. malariae. 11 to 12 days for P. knowlesi. Symptoms can appear in 7 days. Sometimes, the time between exposure and signs of illness may be as long as 8 to 10 months with P. vivax and P. ovale. The incubation period may be longer if you are taking medicine to prevent infection (chemoprophylaxis) or because you have some immunity due to previous infections.

Variation in symptoms
In regions where malaria is present, people who get infected many times may have the disease but have few or no symptoms.Also, how bad malaria symptoms are can vary depending on your general health, what kind of malaria parasite you have, and whether you still have your SPLEEN.

Common symptoms of malaria

In the early stages, malaria symptoms are sometimes similar to those of many other infections caused by bacteria, viruses, or parasites. Symptoms may include: Fever. Chills. Headach. Sweats. Fatigue. Nausea and vomiting. Symptoms may appear in cycles and may come and go at different intensities and for different lengths of time. But,

especially at the beginning of the illness, the symptoms may not follow this typical pattern. The cyclic pattern of malaria symptoms is due to the life cycle of malaria parasites as they develop, reproduce, and are released from the red blood cells and liver cells in the human body. This cycle of symptoms is also one of the major indicators that you are infected with malaria.

Other common symptoms of malaria

Other common symptoms of malaria include: Dry (nonproductive) cough. Muscle and/or back pain. Enlarged spleen. Malarial parasite may also cause spleenomagely.Once in the blood, multiplication of the parasite inside red blood cells (also known as erythrocytes) is responsible for its severity and mortality associated with the disease. After the parasite invades, the red cells undergo profound structural and morphological changes, dramatically altering their physical properties and impairing circulation. In contrast to normal red blood cells, parasitized cells are rigid and adhere to the lining of the blood vessels and other cell types.Due to which there is increase lysis of RBCs causing spleeenomegaly. Infection with the P. falciparum parasite is usually more serious and may become life-threatening.People with severe P. falciparum malaria may develop bleeding problems, shock, liver or kidney failure, central nervous system problems, coma, and can die from the infection or its complications. Cerebral malaria (coma, or altered mental status or seizures) can occur with

severe P. falciparum infection. It is lethal if not treated quickly; even with treatment, about 15%-20% die Some times there are other conditions with symptom similar to a malarial infection. It is important to see to find out the cause of symptoms. Malaria culture is the method to grow malaria parasites outside the body i.e. in an ex vivo environment. Plasmodium falciparum is currently the only human malaria parasite that has been successfully cultured continuously ex vivo. Although attempts for propagation of the parasites outside of humans or animal models reach as far back as 1912, the success of the initial attempts was limited to one or just a few cycles. The first successful continuous culture was established in 1976.Initial hopes that the ex vivo culture would lead quickly to the discovery of a vaccine were premature. However, the development of new drugs was greatly facilitated Infected human red blood cells are incubated in a culture dish or flask at 37C together with a nutrient medium and plasma, serum or serum substitutes.[5] A special feature of the incubation is the special gas mixture of mostly nitrogen (93% nitrogen, 4% carbondioxide, 3% oxygen) allowing the parasites to grow at 37C in a cell incubator.[6] An alternative to gasing the cultures with the exact gas mixture, is the use of a candlejar. The candlejar is an airtight container in which the cultures and a lit candle are placed. The burning candle consumes some of the oxygen and produces carbon dioxide (CO2), which acts as a fire extinguisher. Carbon dioxide content in fresh air varies between 0.036% and 0.039%, at an app. 5% CO2 concentration the candle stops burning. The number of parasites increased by a

factor 5 approximately every 48 hours (= one cycle). The parasitemia can be determined via blood film, to keep it within the wanted limits, the culture can be thinned out with healthy red blood cells.[7] Infected human red blood cells are incubated in a culture dish or flask at 37C together with a nutrient medium and plasma, serum or serum substitutes.[5] A special feature of the incubation is the special gas mixture of mostly nitrogen (93% nitrogen, 4% carbondioxide, 3% oxygen) allowing the parasites to grow at 37C in a cell incubator.[6] An alternative to gasing the cultures with the exact gas mixture, is the use of a candlejar. The candlejar is an airtight container in which the cultures and a lit candle are placed. The burning candle consumes some of the oxygen and produces carbon dioxide (CO2), which acts as a fire extinguisher. Carbon dioxide content in fresh air varies between 0.036% and 0.039%, at an app. 5% CO2 concentration the candle stops burning. The number of parasites increased by a factor 5 approximately every 48 hours (= one cycle). The parasitemia can be determined via blood film, to keep it within the wanted limits, the culture can be thinned out with healthy red blood cells.[7]

Concentration of P. falciparum-infected erythrocytes by discontinuous density gradient centrifugation in Percoll.[8] The original method for the successful ex vivo propagation of P. falciparum described culture of the parasite under static conditions (Trager-Jensen method).[3] James B. Jensen joined Tragers laboratory as a post-doctoral fellow in 1976. He decided to employ a candlejar instead of the CO2 incubator. In the summer of 1976 Milton Friedman, a graduate student in the Trager lab who was working in the MRC laboratories in The Gambia, arranged for a sample of human blood infected with P. falciparum to be sent to New York. This was diluted with RPMI 1640 (which turned out to be the best of the commercial media) in Petri dishes, placed in a candlejar and incubated. The line grew very well and became FCR-3/Gambia, one of the most widely used strains. Later, other lines would be established using similar methods and the impact of continuous cultivation of P. falciparum was phenomenal especially for the testing of putative antimalarials and for deciphering its genes. A number

of subsequent reports (from as far back as the early 1980s), showed that cell suspension (using a shaking-incubator) significantly increased culture growth. Continuous agitation has also been shown to improve other parameters of culture growth relevant to researchers, such as the prolongation of culture synchrony after synchronization procedures, and a reduction of the rate of multiple infections.[9] Despite this, the practice of culturing the parasite under static conditions remains widespread. The greatest value of the candlejar method is that it can be used in laboratories almost anywhere in the world where there is an incubator, a candle and a desiccator.[10] Around 60% parasitized cells can be obtained using optimized culturing conditions.[1] Recent studies of P. falciparum isolated directly from infected patients indicate that alternative parasite biological states occur in the natural host that are not observed with ex vivo cultivated parasites.[11] To achieve synchronization and/or concentration of the parasites in culture several methods have been developed. A discontinuous Percoll gradient procedure can be used to isolate Magnetic collection of P.falciparum infected blood[12] infected red blood cells because red cells containing plasmodia are less dense than normal ones. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml.[13] There are studies, however, that suggest that some strains of P.falciparum are affected in their capacity of invasion after being exposed to this chemical. The difference between diamagnetic low-spin

oxyhemoglobin in uninfected red blood cells and paramagnetic hemozoin in infected red blood cells can also be used for isolation. Magnetic columns have shown to be less harmful for the parasite and are simple and adjustable to the needs of the researcher.[14][15] The column is mounted in a potent magnet holder and the culture flowed through it. The column traps the erythrocytes infected with the latest stages of the parasites, which can then be eluted when the column is removed from the magnet. It is a simple method that does not need expensive equipment and it does not seem to affect the parasites as to their invasion capabilities afterwards.