Professor M.A.

Elkadi

Plasmodium diagnosis

The accepted laboratory practice for diagnosis of malaria is by microscopic examination of blood films stained with Giemsa, Wrights or Fields stain. Blood obtained by pricking a finger or earlobe is the ideal sample because the density of trophozoites or schizonts is greater in blood from capillary-rich areas. Blood obtained by venipuncture collected in heparin or Sequestrine (EDTA) anticoagulant-coated tubes is acceptable if used shortly after collection to prevent alteration in the morphology of parasites.

Professor M.A.Elkadi

Plasmodium diagnosis

 Thick blood film concentrates layers of red blood cells on a small surface by a factor of 20 to 30 times more than thin film. Thick film is stained as an unfixed preparation using Fields stain or diluted Wrights or Giemsa stain. It provides enhanced sensitivity and is much better than the thin film for detection of low parasitemia, in recrudescence or relapse. Lysis of RBC during the staining process can make the parasites detected among the WBC and platelets.

Professor M.A.Elkadi

Plasmodium diagnosis

A drop of blood is placed in the middle of a microscope slide. With the corner of a second slide spread the drop until it is about 10-15 mm in diameter. The thickness should be such that it is just possible to see news print through it. Allow the films to dry in fly proof cabinet. Do not fix the sample. The film is de-hemoglobinised in water and stained with giemsa. Rapid giemsa: 10% giemsa in buffered water at pH 7.1. Immerse the slide for 5 minutes, rinse gently for 1-2 seconds in a jar of tap water. Drain, dry and examine. Standard giemsa: 4% giemsa in buffered solution at pH 7.1. Immerse the slide for 30 minutes. Rinse with fresh water, drain and dry.
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Professor M.A.Elkadi

Plasmodium diagnosis

 Examination of thick film has the advantage of concentrating the parasites by 20 fold in comparison to a thin film. The parasites may appear distorted making species identification difficult. The species should be confirmed by thin film examination. Ideally blood should be collected when the patient's temperature is rising.

The expected sensitivity achieved by an experienced microscopist for the examined thick blood film is about 50 parasites/l which is equivalent to 0.001% of RBC infected
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 Thin film examination is the gold standard in diagnosis of malaria. It is a methanol fixed and stained blood film. Giemsa or Wrights stain is used with buffered water at pH 7.2. Because of the fixed monolayer of RBC available in this procedure, morphological identification of parasite species is easier and provides greater specificity than the thick-film examination. Thin blood film is preferred for routine estimation of the parasitemia. The ability to count parasites in sequential blood films enables the response to therapy to be monitored.

Professor M.A.Elkadi

Plasmodium diagnosis

When the film is dry, fix with 1-2 drops of methanol and stain with: Giemsa: cover the film with 10% giemsa and leave for 30 minutes, wash with distilled water, drain, dry and examine. Leishman's stain: add 7-8 drops and leave it for 1-2 minutes then add 12-15 drops of buffered distilled water, mix thoroughly, leave for 4-8 minutes. Wash off with clean water, drain, dry and examine.

Professor M.A.Elkadi

Plasmodium diagnosis

 Merozoites infect young red blood cells with one trophozoite / RBC usually. The infected RBCs are enlarged, lose color and form Shuffners dots. The ring forms tend to be large and coarse occupying about one third of the infected cell. Trophozoites are generally amoeboid with large chromatin. The mature schizonts contains 12-24 merozoites. Developing forms are frequently present in peripheral blood. Gametocytes are produced after 4 days.

Professor M.A.Elkadi

Plasmodium diagnosis

Professor M.A.Elkadi

Plasmodium diagnosis

The infected cells are of normal size. Rings appear fine and delicate. Several rings appear in one cell. Some rings may have two chromatin dots and some acquire marginal or appliqu forms. Maurer's dots may be present (larger than Shuffners dots). Gametocytes develop after 10 days and appear in peripheral blood. They have characteristic crescent shape. No other developing forms could be seen in the peripheral blood films. The trophozoites are small, and usually multiple per RBC.

Professor M.A.Elkadi

Plasmodium diagnosis

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Professor M.A.Elkadi

Plasmodium diagnosis

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Plasmodium diagnosis

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The infected red cells are not enlarged. Ring forms may have a squarish appearance Band form trophozoites are characteristic of this species Mature schizonts may have up to ten merozoites. Chromatin dot may be on the inner surface of the ring.
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Professor M.A.Elkadi

It is the rarest form in humans. Some infected erythrocytes are enlarged. Rings are large and coarse. Comet forms are common. Schuffner's dots, may be prominent. Mature schizonts are similar to those of P. malariae but larger and more coarse. Gametocytes appear later than other types (3 weeks)
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Professor M.A.Elkadi

Plasmodium diagnosis

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Plasmodium diagnosis

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Detection of the parasites in thick and thin giemsa-stained blood smears remains as the golden standard. Microscopic blood film examination has qualitative and quantitative features that are not associated with other techniques. With careful examination, thin film can provide clues regarding the degree of parasitemia and presence of pigments in neutrophils and monocytes which carries bad prognosis. In P.falciparum, it is essential to examine both thin and thick films and not to accept the first negative report as final because of sludging of schizonts in deep viscera.
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Professor M.A.Elkadi

 It is labor-intensive, staining process may take up to 60 minutes. Interpretation requires considerable expertise, particularly at low levels of parasitemia. In P.falciparum the parasites are sequestered in the visceral capillaries, and may not be seen in peripheral blood.
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Professor M.A.Elkadi

Plasmodium diagnosis

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 Becton-Dickinson's Quantitative Buffy Coat (QBC) method, involves centrifuging the blood in special capillary tubes pre coated with acridine orange. The parasite DNA will be stained with acridine orange. A small molded plastic float presses the parasitized red cells in the uppermost layer of red cell column against the wall of the tube, where they can be viewed by ultra violet light microscopy. The sensitivity of this method is very high.

Professor M.A.Elkadi

Plasmodium diagnosis

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 1. 2. 3. 4. 5. 6. 7. 8.

A variety of abnormalities may be seen in association with malaria: Normochromic, normocytic anemia Thrombocytopenia Leukocytosis or leukopenia Hypoglycemia Hyponatremia Elevated liver enzymes Renal functions tests abnormalities and proteinuria Patients with complicated malaria show massive intravascular hemolysis with hemoglobinemia and hemoglobinuria

Professor M.A.Elkadi

Plasmodium diagnosis

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 Malaria is associated with a normal or reduced leucocyte numbers, leucocytosis is found in terminal cases. Platelet count is moderately or markedly reduced in about 80% of patients with malaria.
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Plasmodium diagnosis

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 They are techniques based on dipstick format and include ICT-Malaria Pf, Opti MALr and the Kat-Quick kits. These tests use colloidal gold labelled monoclonal antibodies bound to nitrocellulose strips to identify and react with water soluble peptides derived from schizont of P.vivax and P. falciparum. The detectable peptides are either Histidine Rich Protein-2 (HRP-2) or parasite-specific Lactate Dehydrogenase (pLDH) which is a specific product of P. falciparum.

Professor M.A.Elkadi

Plasmodium diagnosis

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 Two dip-stick formats are currently avaialable, using monclonal or polyclonal antibodies: antigen capture and antigen competition. Immunochromatographic (ICT) malaria tests are rapid antigen capture assays based on one step of in-vitro immunochromatographic technology to detect circulating plasmodium antigens in a drop of whole blood.

Professor M.A.Elkadi

Plasmodium diagnosis

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Dipstick tests have the potential of enhancing the speed and accuracy of diagnosis, so very useful screening and confirmatory tools. Sensitivities and specificities are approaching 100% with 6% cross reactivity with rheumatoid factor.
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Professor M.A.Elkadi

 Unable to indicate the parasite load. They are useful additions to the established thick and thin blood films, which are regarded "gold standards. Circulating antigens may be detected after elimination of viable parasites from the circulation, so positive results may not always be due to active infection.

Professor M.A.Elkadi

Plasmodium diagnosis

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Antigens detection in serum means current infection, the ideal target antigen should be:

1. not persistant after disapperance of parasitemia. 2. abundant in different body fluids including urine. 3. specific to avoid cross reactions with other microorganisms or body tissues. 4. easy to assay, robust, inexpensive and quantitative. ELISA, RIA, Immunosorbant, western blotting and Immunochromatrgrphic are used to detect plasmodium antigens.

Professor M.A.Elkadi

Plasmodium diagnosis

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Anti-plasmodial antibodies are measured by IFAT- ELISA- EIARIA. The examined antibodies are against the soluble antigens of erythrocytic stages which appear in the blood few days after infection. Antibobies rise quickly to plateau level, maintained for some time then fall slowly. The intensity of antibodies production depends on: species of parasite previous exposure after cure (titres fall to undetectable levels)
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Professor M.A.Elkadi

Presence of plasmodium in the blood means parasite DNA and RNA molecules. Various methods based on principle of nucleic acid hybridization can detect these molecules. Such methods have their place in quality control checks on microscopic diagnosis and to determine the distribution of drug resistance genes. By PCR, It is possible to detect <10 parasites per 10 ul of blood and PCR may prove valuable addition to blood films in diagnosis and speciation of malaria.

Professor M.A.Elkadi

Plasmodium diagnosis

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 Certain enzymes can be used as bio-markers in diagnosis.

The parasite glutamate dehydrogenase (GDH-NADP+) is an excretion antigen which appears as sensitive marker. It is found in plasma and in P.falciparum culture supernatants.
Immunoabsorbent techniques and western blotting are used to isolate this protein by affinity chromatography using rabbit anti-Proteus spp. and GDH (NADP+) serum as ligand. This technique permits the chromatographic detection of P. falciparum excretion antigen that may be used in the production of monoclonal antibodies to improve immunodiagnostic assays and detection of antigenemia.
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 P.falciparum can be maintained in continuous culture in human erythrocytes incubated at 38C in RPMI 1640 medium with human serum under 7% carbon dioxide and 1-5% oxygen. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours. The media should be changed every 24 h, treated with a protease inhibitor cocktail then stored at -70oC until use.
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Plasmodium diagnosis

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