Examination of blood for parasites Medical Parasitology Examination of blood for parasites

Parasites found in blood Microfilaria. Malaria. Babesia. Lieshmania. Trypanosomes. Toxoplasma.

Preparation of blood films Thin blood film Place a small drop of blood near one end of a clean slide. Spread by another slide held at an angle, so that the blood drop will run along the back of the spreader edge. The spreader slide is then pushed forward to the other end of the slide spreading a thin film of blood. Air dry. Fixation by methyl alcohol. Stain in staining dishes. Wash with distilled water, air dry and examine.

Thick blood film Place 4 drops of blood close together on the centre of a slide. Pool the drops together with the corner of another slide making a square of 1x1 cm. Dehaemoglobinize by immersion in distilled water until Hb dissolves and the film become transulescent. Air dry. Stain in staining dishes. Wash with distilled water, air dry and examine.

BLOOD FILMS WITH GEIMSA Thin Thick Blood drop Circular motion spread Air dry Air dry Fix by methyl alcohol 10-30 sec Dehaemoglobinzed Geimsa stain 45 min Wash & dry Geimsa stain Wash & dry

BLOOD FILMS WITH LIESHMAN’s Thin Thick 3 Blood drops Blood drop Circular motion spread Air dry Air dry Fix and stain 1 min Dehaemoglobinzed Transfere into 1 stain: 3 distilled water. Wash and dry Follow the same as in thin film

Fixation with methanol Fixation with methanol. This step is not needed in Lieshman’s stain as it contains methanol. Staining by immersion in the staining dishes Washing of the thin film Careful washing of the thick film

Normal peripheral blood smear

Thin blood films Thick blood films Mainly with intracorpuscular parasites as Malaria and Babesia for morphological description in relation to RBCs. Thick blood films Thick films allow to screen a larger volume of blood and is better with scanty infections. Mainly with sheathed microfilaria as the sheath is the characteristic for type of parasite may be disturbed by spreading the thin films.

Microfilaria The larval stage of the filarial worms. It is found in blood in cases of W. bancrofti and B. malayi - the causative organisms of lymphatic filariasis- showing periodicity. Lymphatic filariasis is a disease transmitted by bite of female mosquitoes of genus Culex, Aedes and Anopheles. Thick blood film is preferred for examining a blood film for microfilaria. Concentration techniques can be used prior to microscopic examination “Knott’s conc technique”.

KNOTT’S CONC. TECHNIQUE Citrated blood 1 ml 10 ml centrifuge Air dry Geimsa 2 min Formalin 2 % sediment Microfilaria

Thick blood film showing microfilaria of W. bancrofti Thick blood film showing microfilaria of B. malayi

Malaria Four species are considered true parasites of humans, as they utilize humans almost exclusively as a natural intermediate host: P. falciparum, P. vivax, P. ovale  and P. malariae.  It is transmitted through bite of female Anopheles mosquitoes. Microscopic identification by examining thick and thin blood films is the method most frequently used to demonstrate an active infection. In P. vivax, P. ovale  and P. malariae ring, trophoziote, schizont, gametocyte stages can be seen in a blood film. In cases of P. falciparum only ring and gametocyte stages are seen because of the adhesion phenomena.

P. vivax Ring-forms Thick blood film Thin blood film

P. vivax -Schizonts Thin blood film Thick blood film

P. vivax -Macrogametocytes Thick blood film Thin blood film

P. falciparum -Ring-forms Thick blood film Thin blood film

P. falciparum -Gametocytes Thick blood film Thin blood film

Babesia It is a zoonatic disease transmitted to human by tick bite. Microscopic identification by examining thick and thin blood films is the method most frequently used to demonstrate an active infection. B. microti B. divergens

Trypanosomes Trypanosoma brucei A wet blood preparation should be examined for the motile trypanosomes, and in addition a smear should be fixed, stained and examined.  Thin and thick blood stained smears for visualization of parasites. Concentration techniques can be used prior to microscopic examination “Buffy coat film”.  African trypanosomiasis “sleeping sickness” is transmitted by the bite of Tsetse fly.

Trypansoma brucei ssp. in thin blood film Trypansoma brucei ssp. in thick blood film

Trypanosoma cruzi A wet blood preparation should be examined for the motile trypanosomes, and in addition a smear should be fixed, stained and examined.  Concentration techniques can be used prior to microscopic examination “Buffy coat film”.  Thin and thick blood smears stained with Giemsa, for visualization of parasites. American trypanosomiasis “Chaga’s disease” is transmitted by the bite of Rudvid bug.

T. cruzi trypomastigotes in a thick blood smear stained with Giemsa T. cruzi trypomastigotes in thin blood smears stained with Giemsa Note the typical C-shape of the trypomastigote that characterizes T. cruzi in fixed blood smears

Lieshmania Concentration techniques can be used prior to microscopic examination “Buffy coat film”.  Thin and thick blood stained smears stained for visualization of parasites. Leishmania is transmitted by the bite of Sand fly. Leishmania spp. amastigotes

Buffy coat film plasma WBC (BC) centrifuge Air dry Fix 30 min RBC spread Geimsa Tryp., L. donovani Citrated blood

Toxoplasma Thin and thick blood stained smears for visualization of parasites in cases of acute toxoplasmosis. Tachyzoite stage in thick blood film