Plasmodium Knowlsei in Thick and Thin Films

Plasmodium Knowlsei in Thick and Thin Films

Thin Smear: P. Knowlsei Ring-form

Background Information of Plasmodium Vivax

Plasmodium knowlesi is a formerly known as a primate malaria parasite commonly found in Southeast Asia. that inflicts malaria in long-tailed macaques, but unfortunately, their incidence in humans are emerging to become the sixth major human malaria parasite (following the division of Plasmodium ovale into 2 species). The transmission of this malarial parasite via Anopheles mosquito may cause severe malaria as indicated by its asexual erythrocytic cycle of about 24 hours, along with an associated fever that typically occurs at the same frequency (i.e. the fever is quotidian) It accounts for up to 70% of malaria cases in certain areas in South East Asia where it is commonly found and is the most common cause of malaria in childhood in the Kudat district of Sabah, Malaysia. 

Symptoms typically surface approximately 11 days after an infected mosquito has bitten a host (Incubation time) and the parasites can be seen in the blood between 10 – 12 days after infection. The parasite may then multiply rapidly resulting in very high parasite densities that may be fatal

Cellular Description

The appearance/morphology of P. knowlesi is synonymous to that of P. malariae and is unlikely to be correctly distinguished/diagnosed unless using PCR assay and/or molecular characterization assays are avaialble.

 

P. malariae is characterized by a compact parasite (all stages) and does not alter the host erythrocyte's shape or size or cause enlargement. Elongated trophozoites stretching across the erythrocyte, called band forms, are sometimes observed. Schizonts will typically have 8-10 merozoites that are often arranged in a rosette pattern with a clump of pigment in the center.

Plasmodium Vivax in Thick and Thin Films

Plasmodium Vivax in Thick and Thin Films

 

Thin Smear

Background Information of Plasmodium Vivax

Plasmodium Vivax is the most common and widely distributed protozoal parasite that infects humans, but fortunately it is the less virulent than Plasmodium Falciparum. P. vivax is carried by the female Anopheles mosquito, since it is only the female of the species that bite. The West Africans are almost safe from this infection as their RBCs do not posses Duffy Antigen  which is the mode of RBC infection. It has an incubation period of 10-17 days and a periodicity of 48 hours

Cellular Description

Schüffner's dots in Thin Smear

 

The hall mark of Plasmodium Vivax infection is enlarged RBCs that maybe round/oval  but often adopting bizarre shapes due to parasitic activity. Presence of Schüffner's dots (golden brown/pinkish-red) scattered through out the RBC cytoplasm is a signature characteristic to this species.  

The parasite within it is often wildly irregular in shape (described as "amoeboid"). Schizonts of P. vivax have ~20 merozoites within them. It is rare to see cells with more than one parasite within them. Merozoites will only attach to immature blood cell (reticulocytes) and therefore it is unusual to see more than 3% of all circulating erythrocytes parasitised.

Plasmodium Falciparum in Thick and Thin Films

Plasmodium Falciparum in Thick and Thin Films

Thick Smear: Maurer's Cleft/Spot

Thin Smear: Maurer's Cleft/Spot

Thin Smear (TOP): Gametocyte
Thick Smear (Bottom): Gametocyte 

Background Information of Plasmodium Vivax

Plasmodium falciparum is the most lethal human protozoan parasite in the Plasmodium species that is responsible in the incidence of malaria and is transmitted by the female Anopheles mosquito. It has the highest rates of complications and mortality with an estimated 247 million human malarial infections (98% in Africa, 70% being 5 years or younger) as of 2006. Crucially, almost every malarial death is caused by P. falciparum infection. The Sub-Saharan Africa is attributed with the highest prevalence. , >75% of cases in most African countries were diagnosed as P. falciparum infection. Fortunately, in other parts of the world with malaria transmission the other less virulent plasmodial species predominate. 

 

It has a periodicity of 36-48 hours and an incubation period of 8-11 days

Cellular Description

 

The hall mark of Plasmodium Falciparum infection is the presence of headphone-like maurer's cleft/spot in RBCs. RBCs in this infection are usually not enlarged. Only young trophozoites and gametocytes can be seen in PBFs.

 

Morphological Characterisitics for the identification of Plasmodium Species

Morphological Characteristics for the identification of Plasmodium Species

Diagrammatic Descriptions for the identification of Plasmodium Species at Trophozoites and Gametocytes Stages

Thick and Thin Smears for Microscopy detection of Malarial Parasites 

Background Information of Thick and Thin Smears

Traditionally the "GOLD-STANDARD" for laboratory diagnosis of malaria has been based on the microscopic examination of a well stained blood film. Both thick and thin peripheral blood films are used for detection and species identification.

[A] Thin Films 

Species identification is more apparent on the thin film because red cell morphology is better preserved than in the thick film. This enables the observer to confirm the diagnosis and identify the species of parasite.

Making Thin Films

1. Transfer a small drop of blood about 1.0 cm from the end of a clean glass slide.
2. Place the slide on a flat surface and hold the long edges between your thumb and forefinger.
3. Using your other hand, place the narrow end of a second (spreader) slide on the specimen slide, just in front of the drop of blood. Hold the spreader at a 30⁰ angle and draw it back until it just touches the drop of blood. Allow the blood to run almost to the edges of the spreader.
4. Push the spreader forward with one light, smooth motion. A thin film of blood in the shape of a bullet or tongue will be formed.

[B] Thick Films

Thick films are extremely useful when parasites are scanty but identification of species may be more difficult than in thin films, especially for P. ovale which is indistinguishable from P. vivax. Thick films should give 10 to 20 times the concentration of thin blood films. Mixed infections may be missed using thick films as during the process of staining the red cells are completely lysed and parasites may be distorted thus making identification of the species much more difficult. Thick films, if well stained, may also be used to perform the differential white cell count in cases with severe leucopenia.

Making Thick Films

1. Place a drop of blood in the center of a microscope slide and, using the corner of another slide, spread it in a circular manner to cover an area approximately two centimeters in diameter. The correct thickness has been achieved if, when placed over a wristwatch, the figures on the dial are just visible.
2. Allow thick films to air-dry for at least one hour at room temperature or thirty minutes at 37⁰C before staining. If films are not completely dry, or are too thick, they may be lost during the staining process.

Routine Staining procedures using Giemsa Stain

Malarial Parasites

Malarial Parasites

Background Information 

Malaria is one of the most serious health hazards facing mankind today. Each year there are more than 400 million clinical cases around the world and almost 2 million of those infected will die from the disease. Most cases occur in South East Asia, India, Africa and parts of South America. The parasite belongs to the Plasmodium genus and is transmitted by pregnant female Anopheles mosquitoes. 

Types of Plasmodium Species

1. Plasmodium falciparum 
2. Plasmodium vivax
3. Plasmodium ovale
4. Plasmodium malariae
5. Plasmodiun knowlesi 

Life-Cycle of Plasmodium Parasites

When the parasite first enters the human host, following the bite from an infected mosquito, they multiply very quickly in the host's liver before migrating into the blood. More female Anopheles mosquitoes are infected when they feed from the infected person. The parasites multiply in the stomach wall of the mosquito, then migrate through her body and infest the salivary gland. When she bites another victim, the next generation of parasites is injected into their blood stream. The life cycle then continues.

After the human host is infected by the parasite there is a variable incubation period before signs and symptoms develop. The typical symptoms of malaria include fever that occurs at intervals of 3-4 days depending on the species of malaria. Species of Plasmodium parasites vary from each other in their genetic make up and this results in different incubation times. P. falciparum takes about 7-14 days for symptoms to show. P. vivax and P. ovale take up to 8-14 days and P. malariae can take up to 7-30 days to manifest.

Reticulocytes

Reticulocytes