l9- malaria diagnosis

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Professor M.A.Elkadi Plasmodium diagnosis 1


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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 idealsample 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 themorphology of parasites.



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Thick blood film concentrates layers of red blood cells on a smallsurface by a factor of 20 to 30 times more than thin film.

Thick film is stained as an unfixed preparation using Fieldsstain or dilutedWrights or Giemsa stain.

It provides enhanced sensitivity and is much better than the thinfilm for detection of low parasitemia, in recrudescence orrelapse.

Lysis of RBC during the staining process can make the parasitesdetected among the WBC and platelets.



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A drop of blood is placed in the middle of a microscopeslide.

With the corner of a second slide spread the drop until it isabout 10-15 mm in diameter. The thickness should be suchthat 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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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 to0.001% 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 orWrights 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 theparasitemia.

The ability to count parasites in sequential blood films enables theresponse to therapy to be monitored.



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When the film is dry, fix with 1-2 drops of methanol and stainwith:

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, leavefor 4-8 minutes. Wash off with clean water, drain, dry and

examine.



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Merozoites infect young red blood cells with one trophozoite / RBCusually.

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.



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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.



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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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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 ofP. malariae but larger and more

coarse. Gametocytes appear later than other types (3 weeks)



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Detection of the parasites in thick and thin giemsa-stainedblood smears remains as the golden standard.

Microscopic blood film examination has qualitative and

quantitative features that are not associated with othertechniques.

With careful examination, thin film can provide cluesregarding the degree of parasitemia and presence ofpigments 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 asfinal because of sludging of schizonts in deep viscera.



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It is labor-intensive, staining process may take up to 60 minutes.

Interpretation requires considerable expertise, particularly at low levels ofparasitemia.

In P.falciparum the parasites are sequestered in the visceral capillaries,and may not be seen in peripheral blood.



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Becton-Dickinson's Quantitative Buffy Coat (QBC) method, involvescentrifuging the blood in special capillary tubes pre coated with acridineorange. The parasite DNA will be stained with acridine orange.

A small molded plastic float presses the parasitized red cells in theuppermost layer of red cell column against the wall of the tube, where theycan be viewed by ultra violet light microscopy. The sensitivity of thismethod is very high.



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A variety of abnormalities may be seen in association withmalaria:

1. Normochromic, normocytic anemia

2. Thrombocytopenia

3. Leukocytosis or leukopenia

4. Hypoglycemia

5. Hyponatremia

6. Elevated liver enzymes

7. Renal functions tests abnormalities and proteinuria

8. Patients with complicated malaria show massive intravascularhemolysis with hemoglobinemia and hemoglobinuria



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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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They are techniques based on dipstick format and includeICT-Malaria Pf, Opti MALr and the Kat-Quick kits.

These tests use colloidal gold labelled monoclonal antibodiesbound 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 ofP. falciparum.



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Two dip-stick formats are currently avaialable, usingmonclonal or polyclonal antibodies: antigen capture andantigen competition.

Immunochromatographic (ICT) malaria tests are rapid

antigen capture assays based on one step of in-vitroimmunochromatographic technology to detectcirculating plasmodium antigens in a drop of wholeblood.

Professor M.A.Elkadi Plasmodium diagnosis25


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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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Unable to indicate the parasite load. They are useful additions tothe 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 alwaysbe due to active infection.



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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.



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Anti-plasmodial antibodies are measured by IFAT- ELISA- EIA-

RIA.

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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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 onmicroscopic diagnosis and to determine the distribution of drug

resistance genes.

By PCR, It is possible to detect


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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 inP.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 ofP.

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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Professor M A Elkadi Plasmodium diagnosis33

l9- malaria diagnosis

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