Leishmania Parasites

• Leishmania are parasitic to humans and are a type of haemoflagellate.

Leishmania Species

• Leishmania donovani causes visceral leishmaniasis.
• Leishmania tropica causes oriental sore or cutaneous leishmaniasis.
• Leishmania Brasilenses causes espundia or muco-cutaneous leishmaniasis.

Leishmania donovani

• Endemic in many places like India, China, Africa, Southern Europe, South America, and Russia.
• Intracellular parasite that lives in a vertebrate host, specifically humans.
• It occurs in the amastigote form, acting as a parasite of the reticuloendothelial system.
• Morphology and life cycle have two forms: amastigote and promastigote.

Amastigote Form (L.d bodies)

• Ovoid or rounded, 2-4 µm in size and mostly found in macrophages in the spleen, liver, and bone marrow.
• Also found in macrophages, monocytes, neutrophils, or rounded endothelial cells.
• They multiply by binary fission, producing numerous daughter cells which distend and rupture the macrophage, leading to phagocytosis by other macrophages.
• When a sand fly feeds on an infected person, the amastigote enters with the blood meal.
• In the sand fly's stomach, it elongates and develops into the promastigote form.

Promastigote Form

• Long, spindle-shaped cells that are 15-25 µm long.
• They multiply by longitudinal binary fission, forming large rosettes with entangled flagella.
• They migrate from the stomach to the pharynx, accumulate, and block the passage.
• Parasites are deposited, phagocytized by macrophages, change into amastigotes, multiply, and enter the midgut of the sand fly when it bites an infected person.
• Promastigotes in artificial cultures have the same morphology as those in the sand fly.
• Giemsa stain shows pale blue cytoplasm and a red nucleus, with a deeply red-stained, rod-like body called a kinetoplast.

Pathogenicity

• The incubation period is usually 3 to 6 months, but may range from 10 days to two years.
• Infection with L. donovani causes Kala-azar or visceral leishmaniasis.
• Characterized by pyrexia, splenic enlargement, liver enlargement, and lymphadenopathy.

Diagnosis

• Specimens are obtained from bone marrow smear, splenic aspirate, and histological sections of biopsy samples from the spleen, liver, and lymph nodes.
• Giemsa stain provides sharp staining results.
• Parasites may be found in peripheral blood smears if the infection is severe.

Serological Test

• Aldehyde (formal gel) test, also known as the Napier test, depends on the increase of serum gammaglobulin.
• 1 ml of patient serum is mixed with a drop or two of 40% formalin in a small test tube.
• A positive result is indicated by the jellification of the mixture like the white of a hard-boiled egg within 3 to 30 minutes.
• Approximately 85% of patients with the disease for 4 months or more show a positive reaction.

Culture

• Aspirated material can be cultured on N.N.N blood agar medium.
• Incubation at 22-24°C leads to promastigote forms appearing in about 10 days.

Leishmania Tropica

• This parasite causes cutaneous leishmaniasis or oriental sore.
• Has an incubation period of 2-8 months.
• Painless ulcers develop at the bite site (oriental sore), usually self-healing after 2 years.
• Sores are distributed on exposed body parts, primarily the face and extremities.
• The parasite is found in the ulcer along the red area with a clean cut margin surrounded by a red areola.

Leishmania Tropica Laboratory Diagnosis

• Smears are made from material obtained from the indurated edge of the sore and stained by Leishman's method.
• Amastigote forms are found in large numbers inside macrophage cells.
• The formal gel test returns negative in cases of cutaneous leishmaniasis.
• Reservoirs of infection include dogs and rodents in forest areas.

Leishmania Tropica Prophylaxis

• Eliminate the reservoir host if possible.
• Control sand flies.
• Protection from sand fly bites via mosquito nets (22 meshes to square inch).

Leishmania Brasilenses

• This parasite causes espundia or muco-cutaneous leishmaniasis.
• Limited to Central and South America.
• Morphological and life cycle are similar to L. donovani and L. tropica.
• Causes skin lesions similar to L. tropica and L. brasiliensis.
• Has an incubation period of a few days to a few weeks.

Malaria Parasites

• Malaria is the most important parasitic disease.
• Classified under Subphylum Sporozoa, Class Telespora, and Genus Plasmodium.
• Geographically, affects a large part of the world from 60N to 40S.

Habitat

• Malaria parasites infect humans and reside inside RBCs after a developmental phase in the liver's parenchyma cells.
• They are carried by the circulating blood to all organs.

Pathogenic Plasmodia

• Four species of plasmodia cause malaria in humans: Plasmodium vivax, P. falciparum, P. malariae, and P. ovale.

Life Cycle and Morphology

• There are two stages during its life cycle: asexual occurring in humans and sexual occurring in the mosquito.

Asexual Phase

• Involves multiplication by division, or schizogony, in two locations: red blood cells (erythrocyte schizogony) and liver cells (exo-erythrocyte schizogony or tissue phase).
• Liver schizogony is essential before the parasite invades RBCs, resulting in the production of merozoites.
• Humans are the intermediate host, with the human phase consisting of asexual multiplication.

Sexual Phase

• Occurs in the female anopheles mosquito, although the sexual forms originate in human RBCs.
• Maturation and fertilization occur in the mosquito, producing numerous sporozoites (sporogony).
• The life cycle of malaria parasites involves alternating asexual and sexual generations.

Human Phase

• Sporozoites, the infective stage, are present in the salivary glands of the anopheles mosquito.
• During a blood meal, the sporozoites pass into the bloodstream.
• Some sporozoites reach the liver and enter the parenchyma cells (hepatocytes), while many are destroyed by phagocytes.

Exo-erythrocytic (tissue) stage

• Within an hour, sporozoites disappear from circulation and start pre-erythrocytic schizogony in the liver.
• Sporozoites enlarge and become rounded inside the liver cells, undergoing repeated nuclear division.
• Each daughter nucleus is surrounded by cytoplasm.
• This stage is called the pre-erythrocytic or primary exo-erythrocytic schizont cycle.
• There is no pigment present in the liver.
• In 6-16 days, the schizont matures and bursts, releasing thousands of merozoites.
• Pre-erythrocytic schizogony lasts approximately 8 days in P. vivax, 6 days in P. falciparum, 9 days in P. ovale, and 15 days in P. malariae.
• Schizogony in the liver involves a small proportion of liver cells, and produces no significant damage or clinical illness.

Hypnozoites

• In P. vivax and P. ovale, some sporozoites entering hepatocytes round up and remain dormant, forming hypnozoites.
• Periodically, some hypnozoites activate, turn into schizonts, and release merozoites, infecting RBCs and causing clinical relapses.

Erythrocytic Stage

• Merozoites released by the pre-erythrocytic schizont invade the RBCs
• Merozoites are pear-shaped bodies, ~1.5µm size
• The entry into the red blood cell only takes about 30 seconds.
• A merozoite rounds up inside of a red blood cell and loses its internal organelles
• In the erythrocyte, having a vacuole in the center, the parasite appears as a rounded body, .
• The cytoplasm is pushed toward its periphery
• Its nucleus, unstained by Giemsa stain, rests at one pole of the cell
• This young parasite form is known as a "ring form".
• Trophozoites feed on haemoglobin of RBCs, but do not metabolize it completely.
• They leave a residue of hemoglobin pigment, called the malaria pigment.
• Upon development, the ring form enlarges and becomes irregular, displaying amoeboid motility (also called an amoeboid form).
• A parasite within an erythrocyte, up until its nucleus starts dividing, is called the TROPHOZOITE.
• Once the nucleus starts dividing, it is then referred to as SCHIZONT. The nucleus divides into diverse amounts of small nuclei surrounded by cytoplasm Merozoite is released by the schizont burst being released into the circulatory system.
• During schizont bursting, released malaria pigment gets phagocytized and observed as pigment granules inside of polymorphs and macrophages.
• The Merozoite then goes on to invade fresh RBCs and repeats development processes.
• Erythrocytic schizogony is repeated in sequential order
• This can lead to intensifying parasitemia until immune response is developed in the respective host.
• Mature schizonts release large quantities of pyrogens upon rupturing
• Fever is due to the work of pyrogens
• Each species has its own duration/time for the erythrocytic schizogony phase
• P. vivax, P. falciparum & P. ovale: 48 hours
• P. malariae: 72 hours

Gametogony

• Following several erythrocyte schizogony cycles, distinct merozoites go on to bypass becoming schizont and begin to develop their sex cells, known as the GAMETOCYTES.
• Sex cell (gametocyte) development occurs in bone marrow, spleen and mature mature gametocytes appear in the peripheral blood stream
• The gametocytes grow in size till nearly filling entirety of the RBC
• The p. falciparum mature gametocytes are crescent shaped, while mature gametocytes tend to be round in shape
• The female or macro gametocyte stains a dark blue in the cytoplasm and has smaller compact red colored nucleus
• Male or micro gametocyte is stained pink or pale blue and has a larger lighter colored diffuse nucleus with prominent pigment granules
• Usually, the females tend to be more numerous than males.
• Gametocytes don't cause clinical illness in the host, and serve to only transmit the infection
• Female gametocytes are known to die within a few days unless consumed by a mosquito.

Mosquito Phase

• Female anopheles mosquitoes will digest non-parasitized erythrocytes during blood meals
• Sexual gametocytes will be set free in the mosquito's stomach
• Microgametocytes divide to form 5 to 8 mobile, whip-like filaments from unique nuclei
• Microgametes/Filaments (male gametes) will lash around for a given amount of time until breaking free
• Male gametes stem from gametocyte, which is a process called ex-flagellation
• Macrogametocytes mature into female gametes, which are not the result of division
• A zygote is formed through the fertilization produced from macrogametes (flagellum)
• Fertilization happens between the duration of one to two hours following eating /taking blood meal
• Ookinete causes zygotes to elongate which creates a vermicular motile shape with apical complex priorly, The Ookinete works to penetrate the mosquito's stomach lining that borders in its respective basement membrane.
• Ookinete matures into rounded shape with an elastic membrane.
• This rounded stage is now named as the Oocyst. Oocyst mature and grows in size, while each nucleus continue their division.
• Within the Oocyst, several thousand of sporozoites go on to develop (sporogony), measuring 10 - 15 µm length with a central nucleus.
• The bulges in the haemocoel are due to those found in the Oocyst (measuring at 50µm) and sporozoites enters it with its ruptures.
• The salivary gland in the haemocoel is where Sporozoites can enter through its ducts. The mosquito can then carry that infection when feeding, as sporozoites are then injected within capillaries near skin when consuming and initiating infections in people.

Pathogenesis and Clinical Picture

• Typical malaria consists of periodic fever, rigged with chills, followed by splenomegaly, and anemia.
• Febrile paroxysm is characterized by the following three continuous stages:

The Cold stage

• Lasts about 15 - 60minute
• The host/patient shivers uncontrollably and has immense feelings of cold

The Hot stage

• Lasts around 2–6 hours
• The host/patient feels intensely hot
• A fever that mounts to 40C or higher
• Severe headache and severe nausea can occur.

The Sweating stage

• The host/patient then sweats profusely The host/patient's temperature begins quickly dropping, leading them into a deep sleep with eventual waking up feeling refreshed
• The paroxysm takes place usually in the early afternoon and can last nearly 8–12 hours
• Clinical manifestation in those with malaria may be due to their own reactions to erythrocytes.

Relapses In Malaria

Due to the formation of hypnozoites from within liver cells that are being reactivated for lengthy periods, the cases of malaria come from P. vivax and P. ovale and usually spans periods of 24 weeks up to 5 years.

Malignant Tertian Malaria

The main form of malaria comes from P. falciparum cases, that if left untreated and without proper care, may develop precarious conditions and is deadly.

Cerebral malaria

• Where Parasitized RBCs may become sticky and cause blockage of vessels within internal organs in the brain That characterized by: hyperpyrexia, coma and paralysis.

Algid Malaria

Surgical complications arise through hypotension through cold/clammy extremities The patient shock resembles symptoms of:

• vomiting
• dysenteric diarrheas
• choleric diarrhea

Black Water Fever

Some patients who went through quinine with improper treatments may include manifestations like:

• dark red or bleak urine passage (also known as black water)
• bilious vomiting
• prostration Auto antibodies due to caused hemolysis leading to above symptoms and the liver can congest with enlargement.

Merozoite-Induced Malaria

• Natural malaria is sporozoite-induced as well. Clinical manifestations are due to being caused by direct RBC infection with schizogony through mosquito injections of merozoite if this same individual has existing erythrocyte activity. The causes for that reaction are
• Receiving blood transfusion from already infected donors
• Blood samples that are shared to others (drug addicts or organ transplant)
• Congenital conditions (mothers passing onto fetus)
• In certain cases, therapeutic- malaria was previously used as syphilis treatments

Biological resistance against malaria

Red cell lineage and resistant genus contribute to developing malarial resistance Hemoglobin deficiency also contributes similar resistance Genus deficiency, malnutrition and deficiencies can provide some needed resistances from contracting malaria.

Laboratory Diagnosis

Diagnosis of malaria can be extracted by inspecting the particular form & gametocyte. A blood smear should be collected a few hours on the peak hours after fever Blood smears must be taken when peak hours ends when parasite are more visible If consecutive smears are negative, then repeat and test more to make way of confirmation either way for that results

Thick film

Make it be by following

• Good amount of blood exude from pricking fingertips with clean spirit, wait to dry, then puncture
• Using slide, patch a square to round spot of moderate blood sample on the slide, then use the horizontal position for even drying, then protect the spot of the the substance with petri dish.

Thin film

Make it by the following

• Put thin film of blood sample on the surface of slide
• Use spreader to firmly and evenly spread the sample to make thin coat - Make sure to use edge to spread blood thinly and properly

The proper way of doing thin film smears is by showing a single unbroken red blood cell, stopping within in a tail formation - Do for roughly 30 seconds, methanol helps fixed for the rest of these thin layer, the same cannot be applied to the thick film

• Add diluted Giemsa stain to the finished thick/thin slides to show proper views of both for around 1-2 hours.
• Finally, proceed to wash and dry slides, then use the oil immersion microscope for review

Serological test

• Immune-precipitation tests
• Immune-fluorescence tests
• Indirect haemagglutination tests
• ELISA tests
• Radio-immunoassay tests
• Using nets for beds and homes
• Destroy breeding and living habitats
• Sprays to kill and remove mosquitos that are vectored Using therapeutic drug for prevention or treat
• Using therapeutic drug for prevention or treat