L9- Malaria Diagnosis
-
Upload
siti-zulaiha-uyub -
Category
Documents
-
view
241 -
download
0
Transcript of L9- Malaria Diagnosis
-
8/3/2019 L9- Malaria Diagnosis
1/33
Professor M.A.Elkadi Plasmodium diagnosis 1
-
8/3/2019 L9- Malaria Diagnosis
2/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 2
-
8/3/2019 L9- Malaria Diagnosis
3/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 3
-
8/3/2019 L9- Malaria Diagnosis
4/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 4
-
8/3/2019 L9- Malaria Diagnosis
5/33
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
Professor M.A.Elkadi Plasmodium diagnosis 5
-
8/3/2019 L9- Malaria Diagnosis
6/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 6
-
8/3/2019 L9- Malaria Diagnosis
7/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 7
-
8/3/2019 L9- Malaria Diagnosis
8/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 8
-
8/3/2019 L9- Malaria Diagnosis
9/33
Professor M.A.Elkadi Plasmodium diagnosis 9
-
8/3/2019 L9- Malaria Diagnosis
10/33
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 10
-
8/3/2019 L9- Malaria Diagnosis
11/33
Professor M.A.Elkadi Plasmodium diagnosis 11
-
8/3/2019 L9- Malaria Diagnosis
12/33
Professor M.A.Elkadi Plasmodium diagnosis 12
-
8/3/2019 L9- Malaria Diagnosis
13/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 13
-
8/3/2019 L9- Malaria Diagnosis
14/33
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)
Professor M.A.Elkadi Plasmodium diagnosis 14
-
8/3/2019 L9- Malaria Diagnosis
15/33
Professor M.A.Elkadi Plasmodium diagnosis 15
-
8/3/2019 L9- Malaria Diagnosis
16/33
Professor M.A.Elkadi Plasmodium diagnosis 16
-
8/3/2019 L9- Malaria Diagnosis
17/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 17
-
8/3/2019 L9- Malaria Diagnosis
18/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 18
-
8/3/2019 L9- Malaria Diagnosis
19/33
Professor M.A.Elkadi Plasmodium diagnosis 19
-
8/3/2019 L9- Malaria Diagnosis
20/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 20
-
8/3/2019 L9- Malaria Diagnosis
21/33
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
Professor M.A.Elkadi Plasmodium diagnosis 21
-
8/3/2019 L9- Malaria Diagnosis
22/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 22
-
8/3/2019 L9- Malaria Diagnosis
23/33
Professor M.A.Elkadi Plasmodium diagnosis 23
-
8/3/2019 L9- Malaria Diagnosis
24/33
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.
Professor M.A.Elkadi Plasmodium diagnosis 24
-
8/3/2019 L9- Malaria Diagnosis
25/33
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
-
8/3/2019 L9- Malaria Diagnosis
26/33
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.
Professor M.A.Elkadi Plasmodium diagnosis26
-
8/3/2019 L9- Malaria Diagnosis
27/33
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.
Professor M.A.Elkadi Plasmodium diagnosis27
-
8/3/2019 L9- Malaria Diagnosis
28/33
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 diagnosis28
-
8/3/2019 L9- Malaria Diagnosis
29/33
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)
Professor M.A.Elkadi Plasmodium diagnosis29
-
8/3/2019 L9- Malaria Diagnosis
30/33
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
-
8/3/2019 L9- Malaria Diagnosis
31/33
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.
Professor M.A.Elkadi Plasmodium diagnosis31
-
8/3/2019 L9- Malaria Diagnosis
32/33
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.
Professor M.A.Elkadi Plasmodium diagnosis32
-
8/3/2019 L9- Malaria Diagnosis
33/33
Professor M A Elkadi Plasmodium diagnosis33