Diagnosis of malaria involves examining thick and thin blood films under a microscope to look for malaria parasites. Thick films are used to detect parasites while thin films allow for species differentiation. Common stains include Giemsa stain which demonstrates parasites clearly. Parasite morphology, stage of infection, species, number and other characteristics are reported. Rapid diagnostic tests can also detect parasite antigens but not mixed infections. Molecular tests can identify species and drug resistance but are not used for clinical diagnosis. Differentiating species involves examining features of infected red blood cells and parasite morphology.

1. Diagnosis of Malaria

2. Diagnosis of malaria
Parasitological diagnosis:
To demonstrate the parasite in thick and thin film:
 Thick film : is more than one layers of blood cells so it can detect malaria
parasite. Blood taken in thick film about 3 drops (0.05m)
 Thin film: is one layer of cells, is used to differentiate the species about one
drop.
* For routine malaria microscope both thick and thin film are made on one
slide .
*Both preferably prepared in one slide and stained with
Giemsa’s.

3. Techniques :
• Finger prick , the 3rd or 4th fingers are used on the lateral
terminal side , in children big toe is used
• Clean the area first then prick, remove the first drop, then 3
drops are taken on one side, then thick film is done in circular
manner by anther slide. One drop of blood is taken for thin film
above the thick film. Thin film is fix by alcohol

4. Staining:
Many stains are used, alcoholic and watery based stains:
 Field stain A & B (watery) for thick film, modified Field stain used
for thin blood film
 Leischman stain (alcoholic) for thin film.
 Giemsa stain, used for both, it is recommended by (WHO), because
demonstrate the parasite very clear.
The chromatin appear deeply reddish. The cytoplasm blue and
granules are pinkish.

6. Characters to look for in Thin B.film.
Parasite:
Stage.
Size.
Shape.
Number.
Accumulation of MP
Cytoplasm: chromatin
Infected RBCs:
Size
Shape
Stippling, clefts, dots…

8. Immunological based test:
• Antigen detection:
Qualitative (rapid diagnostic tests RDTs)
Immune stain (immuno- fluorescent)
• Antibodies detection:
Quantitative (ELISA:)

9. Rapid Diagnostic tests (RDTs)
• Immunologic assays to detect specific antigens
• Commercial kits now available as immunochromatographic rapid
diagnostic tests (RDTs), used with blood
• P. falciparum histidine-rich protein 2 (PfHRP-2)
• parasite LDH (pLDH)
• Monoclonal and polyclonal antibodies used in antigen (Ag) capture test
• Cannot detect mixed infections
• Cross reactivity with rheumatoid factor reportedly corrected

11. Molecular biology:
• (detection of parasite nucleic acid: DNA & RNA) using
techniques like (PCR, RFLP, …)
• Purposes:
• Detection & identification of parasite
• Further characterization down spp.
• Rapid detection of drug resistant strains
• Limitations: Are not suitable for clinical diagnosis

12. Reporting of result:
In the result form the following should be right clearly.
1. Malaria positive or negative.
2. Stages of the parasite .
3. Plasmodium spp detected.
4. Parasite count (density) .
All these are very important in malaria treatment and
follow up , e.g hypeparasitmia needs special treatment.

13. How can we do parasite count (density) ?:
1. Plus system, we count the number of parasite in 100 fields of TK BF, if:
• (1-10) parasites per 100 field (+)
• (1-100) parasites per 100 field (++)
• (1-10) parasites per 1 field (+++)
• more than 10 per 1 field (++++)
2. Parasites number against WBCS:
• It estimate parasites numbers /µL.
• In thick film you count the parasites in 100 fields . At the same time you count WBCs
numbers then you count as follow:
• The parasites No. X 8000(WBCs) = parasites No./µL blood
(WBCs) in 100 fields

14. 3. percentage of infected RBCs:
• Intensity of parasiteamia using TN BF to count at least 300
RBCs.
• Then calculate the percentage of infected RBCs.
4. QBC method:
• AO-coated capillary is filled with 50-100 µl blood.
• Parasites concentrate below the granulocyte layer in tube.
• Fluorescent microscopy after centrifugation.
• May be slightly more sensitive than light microscopy.

15. Differential Daiagnosis of Malaria
Parasites

18. • On blood film we see all the stages of the parasite in P.V, P.O,
P.M, but in P.F we see only the ring forms and gametocytes.
• The ring form in P.F is very small with double chromatin(more
common) and double infection in one RBCs, Merro’s dots are
seen in sever Malaria
• In P.V RBCs are enlarged and the trophozoite is embodied
form,with Shuffeners dots
• In P.O the 30 % of RBCs are ovale in shape, with Jam’s dots.
• In P.M the RBCs normal in shape and size and the trophpzoite is
band form, with Zimman’s dots and a lot of pigments.

19. Species Differentiation on Thin Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoites
Schizonts
Gametocytes

20. P.F hyper parasitaemia in TK BF

22. P. falciparum
One
erythrocytic
cycle = 48 hr
See the sexual
stages
(gametocytes
shape)
Number of
merozoite 14-32

25. P.F Schizonts: 8-32 merozoites
(rarely seen in peripheral blood)

26. P.F gametocytes

27. Plasmodium vivax

32. Plasmodium vivax
early trophozoites
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
Trophozoites: ameboid; deforms the erythrocyte

33. P.V Schizonts: 12-24 merozoites

34. Gametocytes: round-oval

36. Plasmodium ovale
Infected erythrocytes: moderately fimbriated; oval; (Jam’s dots)
Rings

37. P.O Trophozoites: compact

38. P.O Schizonts: 6-14 merozoites;

39. P.O Gametocytes: round-oval

41. Infected erythrocytes: size normal to decreased (3/4X)
Plasmodium malariae
Trophozoite: compact
Early trophozoite: bird eye

42. P.M Trophozoite: typical band form

43. P.M Schizont: 6-12 merozoites;
coarse, dark pigment

44. Gametocyte: round; coarse,
dark pigment