MALARIAAssignment on

BCH 514 Clinical Biochemistry

Submitted bySelma Abdul Samad

BCH-10-05-02S3 MSc.Biochemistry

What is Malaria ??An infectious tropical Disease caused by the parasite Plasmodium sp. in humansThe name malaria derived from the Italian mal’aria or bad airFifth cause of death from infectious diseases worldwide (after respiratory infections, HIV/AIDS, diarrhoeal diseases, and tuberculosis) and the second in Africa, after HIV/AIDSIt is a disease that can be treated in just 48 hours, yet it can cause fatal complications if the diagnosis and treatment are delayed.

Geographical Distribution

#1 priority tropical disease of WHOPrevalent in 108 countries of the tropical and semitropical world (home to half of the world)Every year, malaria is reported to cause more than 250-660 million infections and more than a million deaths (mostly among African children)There has been a sharp decline in the incidence of malaria in South-East Asia since 1977 reflecting a rapid fall in India and Sri Lanka but . there has been a slow increase in South and Central America.

Geographical Distribution

In India .. Orissa, Chhattisgarh, West Bengal, Jharkhand and Karnataka contribute the most number of cases of malaria in India

HISTORY OF MALARIAOne of the oldest diseases known to mankindWars , kings , dynasties etc influencedMalaria was linked with poisonous vapours of swamps or stagnant water on the ground since time immemorial.The word was introduced to English by Horace Walpole, who wrote in 1740 about a “horrid thing called mal’aria, that comes to Rome every summer and kills one.” The term malaria, without the apostrophe, evolved into the name of the disease only in the 20th . century.

Man and Malaria seem to have evolved togetherIt is believed that most, if not all, of today's populations of human malaria may have had their origin in West Africa (P. falciparum) and West and Central Africa (P. vivax) on the basis of the presence of homozygous alleles for hemoglobin C and RBC Duffy negativity that confer protection against P. falciparum and P. vivax respectively

Different studies have suggested that P. falciparum malaria probably jumped from great apes to man, probably by a single host transfer by vector mosquitoesP. malariae, P. ovale, and P. vivax diverged over 100 million years ago along the lineage of the mammalian malaria parasitesP. ovale is the the sole known surviving representative of its line and causes infection only in humans

Famous victims of malaria

Alexander the Great is believed to have died of malaria in 323 BC, on the route to India beyond MesopotamiaGeorge Washington, (1st President of US, 1789-1797): Developed his first bout with malaria in Virginia in 1749 at age 17. He had periodic attacks, recorded in 1752, 1761, 1784, and 1798.Abraham Lincoln (16th President, 1861-1865) had periodic bouts of malaria when growing upJohn F. Kennedy (35th President, 1961-1963) acquired malaria during World War II, about 1943Mother Teresa was hospitalized with malaria in 1993, ……………….and many many others………………..

Scientific Discoveries… Hippocrates was probably the the first malariologist The Hippocratic corpus was the first document to mention about splenic change in malaria and also it attributed malaria to ingestion of stagnant water

Time Line For Scientific Discoveries

Ancient Times - Early man attributed the fevers to evil spirits, angered deities, demons, or the black magic of sorcerers

Severel thousand years ago - Babylonian cuneiform script attributes malaria to a god, pictured as a mosquito-like insect

800 BC - Indian sage Dhanvantari wrote that bites of mosquitoes could causes diseases, fever, shivering etc.

400 BC - Hippocrates described the various malaria fevers of man; distinguished the intermittent malarial fever from the other continuous fevers; noted the daily, every-other-day, and every-third-day temperature rise; mentioned about splenic change in malaria; attributed malaria to ingestion of stagnant water; also related the fever to the time of the year and to where the patients lived

Several theories and hypotheses came for yearsIt was finally in 1884 that Plasmodium was discovered by Russian Scientists and their link with malaria was confirmed by Louis Pasteur.In 1897 Ronald Ross demonstrated oocysts in the gut of anopheline mosquito at Secunderabad, India, proving that mosquito was the vector for malaria

Nobel Prizes for Malaria Related Research

Ronald Ross, 1902: "For his work on malaria, by which he has shown how it enters the organism and thereby has laid the foundation for successful research on this disease and methods of combating it". Ronald Ross demonstrated the oocyst of malarial parasite in the gut wall of a mosquito on August 20, 1897 in Secunderabad, India.Alphonse Laveran, 1907: "In recognition of his work on the role played by protozoa in causing diseases". Laveran was the first to notice parasites in the blood of a patient suffering from malaria on November 6, 1880 at Constantine, Algeria.

Julius Wagner-Jauregg, 1927: "For his discovery of the therapeutic value of malaria inoculation in the treatment of dementia paralytica".

Paul Hermann Müller, 1948: "For his discovery of the high efficiency of DDT as a contact poison against several arthropods".

Etiology5 species of malaria parasites that infect man identified Plasmodium vivax (vivax malaria,

benign tertian malaria) Plasmodium ovale (ovale malaria, ovale

tertian malaria) Plasmodium malariae (quartan malaria) Plasmodium falciparum (falciparum

malaria, malignant tertian malaria, pernicious malaria, subtertian malaria)

Plasmodium knowlesi

The Malaria ParasitesProtozoan parasites called PlasmodiaPhylum: Protozoa Class: Sporozoa Genus: Plasmodia Species : several species, 5 known to affect

man

INFECTIONTRANSMISSION Principal mode of spread of malaria is by

the bites of female Anopheles mosquito Of more than 480 species ofAnopheles,

only about 50 species transmit malaria The habits of most of the anopheline

mosquitoes have been characterised as anthropophilic (prefer human blood meal), endophagic (bite indoors), and nocturnal (bite at night) with peak biting at midnight, between 11 pm and 2 am.

The blood meal from a vertebrate host is essential for the female mosquitoes to nourish their eggs.

The mosquitoes find their host by seeking visual, thermal, and olfactory stimuli and of these, carbon dioxide, lactic acid, skin temperature, and moisture are more important mosquito attractants

When a mosquito bites an infected individual, it sucks the gametocytes, the sexual forms of the parasite, along with blood. These gametocytes continue the sexual phase of the cycle within the mosquito gut and the sporozoites that develop then fill the salivary glands of the infested mosquito. When this female mosquito bites another man for a blood meal, the sporozoites are inoculated into the blood stream of the fresh victim, thus spreading the infection.

Anopheles MosquitoMalaria is transmitted from man to man by the female anopheles mosquitoNearly 45 species of the mosquito have been found in IndiaHow does a mosquito bite?Anopheles Mosquito

Other modes of transmission Mother to the growing

fetus (Congenital malaria) - transplacentally or during labor

Transfusion Malaria: transmitted by transfusion of blood from infected donors.

Needle stick injury: accidentally among health care or due to needle sharing among drug addicts

LIFE CYCLEThe malaria parasite has a complex, multistage life cycle occurring within two living beings, the vector mosquitoes and the vertebrate hosts.Consits of two phases Sexual phase ( sporogony) – in female

anopheles mosquito Asexual phase ( schizogony) – in man

 The parasite passes through several stages of development such as the sporozoites (Gr. Sporos = seeds; the infectious form injected by the

mosquito) merozoites (Gr. Meros = piece; the stage invading the erythrocytes) trophozoites (Gr. Trophes = nourishment; the form multiplying in

erythrocytes) and gametocytes (sexual stages)

all these stages have their own unique shapes and structures and protein complements.The surface proteins and metabolic pathways keep changing during these different stages, that help the parasite to evade the immune clearance, while also creating problems for the development of drugs and vaccines

Sporogony Within the Mosquitoes

Mosquitoes are the definitive hosts for the malaria parasites, wherein the sexual phase(sporogony) of the parasite's life cycle occursResults in the development of innumerable infecting forms of the parasite within the mosquito that induce disease in the human host following their injection with the mosquito biteInfected individual mosquito draws blood the male and female gametocytes of the parasite find their way into the gut of the mosquitoFemale forms macrogametes ; Males form microgamete

The male and female gametes fuse in the mosquito gut to form zygotes, which subsequently develop into actively moving ookinetes that burrow into the mosquito midgut wall to develop into oocysts.Growth and division of each oocyst produces thousands of active haploid forms called sporozoites.After the sporogonic phase of 8–15 days, the oocyst bursts and releases sporozoites into the body cavity of the mosquito, from where they travel to and invade the mosquito salivary glands.

When the mosquito thus loaded with sporozoites takes another blood meal, the sporozoites get injected from its salivary glands into the human bloodstream, causing malaria infection in the human host.It has been found that the infected mosquito and the parasite mutually benefit each other and thereby promote transmission of the infection. The Plasmodium-infected mosquitoes have a better survival and show an increased rate of blood-feeding, particularly from an infected host.The duration of the cycle in the mosquito is known as the external incubation period and varies from 8-10 days (28`C) to 16 days (20`C)

Malaria oocysts on stomach of mosquito

Schizogony in the Human Host

Man is the intermediate host for malaria, wherein the asexual phase of the life cycle occurs.The sporozoites inoculated by the infested mosquito initiate this phase of the cycle from the liver, and the latter part continues within the red blood cells, which results in the various clinical manifestations of the disease.Pre-erythrocytic Phase - Schizogony in the Liver: With the mosquito bite, tens to a few hundred invasive

sporozoites are introduced into the skin. Following the intradermal deposition, some sporozoites are

destroyed by the local macrophages, some enter the lymphatics, and some others find a blood vessel.

The sporozoites that enter a lymphatic vessel reach the draining lymph node wherein some of the sporozoites partially develop into exoerythrocytic stages and may also prime the T cells to mount a protective immune response

The sporozoites that find a blood vessel reach the liver within a few hours

 The sporozoites then negotiate through the liver sinusoids, and migrate into a few hepatocytes, and then multiply and grow within parasitophorous vacuoles.

Each sporozoite develop into a schizont containing 10,000–30,000 merozoites (or more in case of P. falciparum.

The growth and development of the parasite in the liver cells is facilitated by a a favorable environment created by the The circumsporozoite protein of the parasite.

The entire pre-erythrocytic phase lasts about 5–16 days depending on the parasite species: • on an average 5-6 days for P. falciparum,• 8 days forP. vivax,•  9 days for P. ovale,•  13 days for P. malariae and• 8-9 days for P. knowlesi.

The pre-erythrocytic phase remains a “silent” phase, with little pathology and no symptoms, as only a few hepatocytes are affected. This phase is also a single cycle, unlike the next, erythrocytic stage, which occurs repeatedly.

The merozoites that develop within the hepatocyte are contained inside host cell-derived vesicles called merosomes that exit the liver intact, thereby protecting the merozoites from phagocytosis by Kupffer cells.

These merozoites are eventually released into the blood stream at the lung capillaries and initiate the blood stage of infection thereon.

In P. vivax and P. ovale malaria, some of the sporozoites may remain dormant for months within the liver. Termed as hypnozoites, these forms develop into schizonts after some latent period, usually of a few weeks to months.

Erythrocytic Schizogony - Centre Stage in Red Cells Red blood cells are the 'centre stage' for the asexual

development of the malaria parasite. Within the red cells, repeated cycles of parasitic

development occur with precise periodicity, and at the end of each cycle, hundreds of fresh daughter parasites are released that invade more number of red cells.

The merozoites released from the liver recognize, attach, and enter the red blood cells (RBCs) by multiple receptor–ligand interactions in as little as 60 seconds. This quick disappearance from the circulation into the red cells minimises the exposure of the antigens on the surface of the parasite, thereby . protecting these parasite forms from the host immune response.

The invasion of the merozoites into the red cells is facilitated by molecular interactions between distinct ligands on the merozoite and host receptors on the erythrocyte membrane. Glycophorin, the major erythrocytic glycoprotein is involved in merozoite invasion.

The more virulent P. falciparum uses several different receptor families and alternate invasion pathways that are highly redundant and hence can invade any red cell while others like P.vivax can invade using one type of receptor.

The process of attachment, invasion, and establishment of the merozoite into the red cell is made possible by the specialized apical secretory organelles of the merozoite, called the micronemes, rhoptries, and dense granules.

The initial interaction between the parasite and the red cell stimulates a rapid “wave” of deformation across the red cell membrane, leading to the formation of a stable parasite–host cell junction.

Following this, the parasite pushes its way through the erythrocyte bilayer with the help of the actin–myosin motor, proteins of the thrombospondin-related anonymous protein family (TRAP) and aldolase, and creates a parasitophorous vacuole to seal itself from the host-cell cytoplasm, thus creating a hospitable environment for its development within the red cell.

At this stage, the parasite appears as an intracellular “ring”.

The ring forms grow in size to trophozoites . Meanwhile, they utilise Hb . ie., the amino acids are utilised for protein bioshynthesis and the toxic heme is detoxified by heme polymerase and sequestered as hemozoin (malaria pigment)

The parasite depends on anaerobic glycolysis for energy The trophozoite multiplies by schizogony dividing into a

number of small merozoites varying with the species to form a mature schizont.

The erythrocytic phase is called schizogonic periodicity

At the end of the cycle,the merozoites are released by rupture of the red cell membrane and enter new red cells, particularly young red cells.

The erythrocytic cycle occurs every• 24 hours in case of P. knowlesi,•  48 h in cases of P. falciparum, P.

vivax and P. ovale and • 72 h in case of P. malariae. 

During each cycle, each merozoite grows and divides within the vacuole into 8–32 (average 10) fresh merozoites, through the stages of ring, trophozoite, and schizont.

Gametogony : A small proportion of asexual parasites do not

undergo schizogony but differentiate into the sexual stage gametocytes.

These male or female gametocytes are extracellular and nonpathogenic and help in transmission of the infection to others through the female anopheline mosquitoes, wherein they continue the sexual phase of the parasite's life cycle.

Gametocytes of P. vivax develop soon after the release of merozoites from the liver, whereas in case of P. falciparum, the gametocytes develop much later with peak densities of the sexual stages typically occurring 1 week after peak asexual stage densities.

PATHOLOGY The most pronounced changes related to malaria involve the blood and the blood-forming system, the spleen and the liver.Secondary changes can occur in all the other major organs, depending on the type and severity of the infection.The pathological changes are more profound and severe in case of P. falciparum malaria. Severe malaria is a complex multisystem disorder with many similarities to sepsis syndromes.

RBC rupture ; cytoadherence and sequestrationAnemiaThrombocytopenia ; Clotting defectsElevated ESRBone marrow may show evidence of dyserythropoeisis, iron sequestration and erythrophagocytosis in the acute phase of falciparum malaria.Splenomegaly ; Splenic ruptureMalarial hepatitis ; Hepatomegalycardiovascular function abnormalities

GI irritation, ischemia, ulcers , necrosisNephritis ; albuminuria ; kidney failure ; edemaCNS manifestations ( in P.falciparum)

All pathological manifestations increased and severe in case of falciparum malaria

CLINICAL FEATURESAll the clinical features of malaria are caused by the erythrocytic schizogony in the bloodTypical features: It includes three stages viz. Cold stage, Hot stage and

Sweating stage The febrile episode starts with shaking chills, usually at

mid-day between 11 a.m. to 12 noon, and this lasts from 15 minutes to 1 hour (the cold stage)

Followed by high grade fever, even reaching above 1060 F, which lasts 2 to 6 hours (the hot stage).

This is followed by profuse sweating and the fever gradually subsides over 2-4 hours. These typical features are seen after the infection gets established for about a week.

In vivax malaria, this typical pattern of fever recurs once every 48 hours and this is called as Benign Tertian malaria. 

Similar pattern may be seen in ovale malaria too (Ovale tertian malaria).

In falciparum infection (Malignant tertian malaria), this pattern may not be seen often and the paroxysms tend to be more frequent (Sub-tertian). 

In P. malariae infection, the relapses occur once every 72 hours and it is called Quartan malaria.

Atypical features: In an endemic area, malaria often presents with atypical manifestations Atypical fever Headache Body ache, back ache and joint pains Dizziness, vertigo Altered behaviour, acute psychosis Altered sensorium Convulsions, coma Cough Breathlessness Chest pain

Acute abdomen Weakness Vomiting and diarrhoea Jaundice Pallor Puffiness of lids Secondary infections Hepatosplenomegaly Combinations of the above

Clinical features suggesting P. falciparum infection: Presence of any of the complications of P.

falciparum malaria viz. altered sensorium; convulsions; coma; jaundice; severe anemia; hypotension; prostration; hyperpyrexia; renal failure etc.

Atypical presentation. Not responding to chloroquine therapy

within 48 hours Recurrence within 2 weeks

DIAGNOSISInvolves identification of malaria parasite or its antigens/products in the blood of the patientThe efficacy of the diagnosis is subject to many factors The different forms of the four malaria species; The different stages of erythrocytic schizogony; The endemicity of different species; The inter-relation between the levels of

transmission, immunity, parasitemia, and the symptom

The problems of recurrent malaria, drug resistance, persisting viable or non-viable parasitemia etc.

The diagnosis of malaria is confirmed by blood tests and can be divided into microscopic and non-microscopic tests.The microscopic tests involve staining and direct visualization of the parasite under the microscope.1. Peripheral smear study2. Quantitative Buffy Coat (QBC) testNon microscopic techiques involves- Rapid Diagnostic tests , PCR assays , Immunofluorescence for detection of plasmodia , ELISA for malaria antigens , Western blotting etc.

Differential diagnosisMalaria can be offered as a differential diagnosis for a big list of diseases. General: All other causes of fever, migraine, sinusitis, tension headache

etc. Respiratory system: Pharyngitis, bronchitis, pneumonia,

bronchopneumonia, pleurisy. Cardiovascular: Acute myocardial infarction, cardiogenic shock, left

ventricular failure, pericarditis Abdominal: Hepatitis, liver abscess, splenitis, splenic abscess, other

causes of splenomegaly, subdiaphragmatic abscess, acute abdomen, cholecystitis, cholangitis, gastroenteritis, amebiasis, appendicitis, etc.

Central nervous system: Acute encephalitis, meningitis, intra-cranial space occupying lesions, stroke, metabolic encephalopathy etc.

Psychiatry: Acute confusional states, acute psychosis, mood disorders Renal: Acute nephritis, nephrotic syndrome, acute renal failure Haematological: All other causes of anemia; blood dyscrasias,

hemoglobinopathies, hemolytic anemias, intra vascular hemolysis, bleeding diathesis, DIC etc.

TREATMENT History of treatment In the ancient times, limb blood-letting, emesis, amputation

and skull operations were tried in the treatment of malarial fever

Artemisinin: from the herb Artemisia annua (sweet wormwood)

cinchona bark : more than 350 years Many drugs were developed to protect the troops from malaria,

particularly during World War II. Chloroquine, Primaquine, Proguanil, amodiaquine and Sulfadoxine/Pyrimethamine were all developed during this time.

Malarone: In 1998 a new drug combination was released in Australia called Malarone. This is a combination of proguanil and atovaquone. Atovaquone became available 1992 and was used with success for the treatment of Pneumocystis carrinii. The synergistic combination with proguanil is found to be an effective antimalarial treatment.

ANTIMALARIALS - Anti malarial drugs can be classified according to anti malarial activity and structure1. According to anti malarial activity: Tissue schizonticides for causal prophylaxis: These drugs

act on the primary tissue forms of the plasmodia which after growth within the liver, initiate the erythrocytic stage. By blocking this stage, further development of the infection can be theoretically prevented. Pyrimethamine and Primaquine have this activity. However since it is impossible to predict the infection before clinical symptoms begin, this mode of therapy is more theoretical than practical.

Tissue schizonticides for preventing relapse: These drugs act on the hypnozoites of P. vivax and P. ovale in the liver that cause relapse of symptoms on reactivation. Primaquine is the prototype drug; pyrimethamine . . also has such activity.

Blood schizonticides: These drugs act on the blood forms of the parasite and thereby terminate clinical attacks of malaria. These are the most important drugs in anti malarial chemotherapy. These include chloroquine, quinine, mefloquine, halofantrine, pyrimethamine, sulfadoxine, sulfones, tetracyclines etc.

Gametocytocides: These drugs destroy the sexual forms of the parasite in the blood and thereby prevent transmission of the infection to the mosquito. Chloroquine and quinine have gametocytocidal activity against P. vivax and P. malariae, but not against P. falciparum. Primaquine has gametocytocidal activity against all plasmodia, including P. falciparum.

Sporontocides: These drugs prevent the development of oocysts in the mosquito and thus ablate the transmission. Primaquine and chloroguanide have this action.

Thus in effect, treatment of malaria would include a blood schizonticide, a gametocytocide and a tissue schizonticide (in case of P. vivax and P. ovale). A combination of chloroquine and primaquine is thus needed in ALL cases of malaria.

Other Drugs for Chemotherapy of MalariaClindamycin: It acts by inhibiting the protein synthesis by binding to the 50s subunit of ribosomes.Fluoroquinolones: Both ciprofloxacin and norfloxacin have been found to have anti malarial activity both in vitro and in vivo. However, results are not consistent.Azithromycin: Azithromycin is found to have anti malarial activity and has been found to be useful as a causal prophylactic agentPyronaridine: Structurally, it resembles amodiaquine and has been found to be highly effective against chloroquine resistant strains in China.Piperaquine: Its activity is similar to that of chloroquine. A combination with artimisinin is undergoing studies.

REFERENCETropical Diseases – Manson Bahr; 19th edition ; 1987 ; page 3-47http://www.malariasite.com/malaria

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