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Laboratory diagnosis of Malaria

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Laboratory diagnosis of Malaria The diagnosis of malaria may in fact into two ways : Direct diagnosis: direct demonstration of the parasite whole cell or of parasite ... – PowerPoint PPT presentation

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Title: Laboratory diagnosis of Malaria


1
Laboratory diagnosis of Malaria
2
  • The diagnosis of malaria may in fact into two
    ways
  • Direct diagnosis direct demonstration of the
    parasite whole cell or of parasites nucleic acid
    or products in the blood
  • Indirect Diagnosis the demonstration of the
    patients immune response to the infection
    (immunodiagnosis).

3
  • (A) Light microscopic observation
  • - Sample preparation
  • 1- thin film observation of malaria
    parasites is optimal when parasites are fixed and
    observed in their natural location within red
    blood cells after appropriate staining. This is
    best accomplished with the thin film preparation
    technique. Unfortunately, thin film has a low
    sensitivity and is thus inadequate for low
    parasitaemic infection.
  • 2- thick film An adequate parasite
    concentration method is obtained by osmotic lysis
    of the red blood cells releasing the parasites,
    as is the case with the thick film preparation
    technique, the sensitivity is more than thin
    film.

4
  • - Fixation
  • Its may be achieved by heat and alcoholic
    solutions for 10-20 seconds . Methanol (methyl
    alcohol) is the most widely used fixative for
    malaria thin films.
  • - staining
  • 1- Giemsa staining procedure
  • Is the most commonly used method for both
    thin and thick films all over the world for the
    quality of the stain and, of greater importance,
    because its stability in tropical climates.
  • 2- Field staining procedure
  • 3- Leishman staining procedure

5
  • Blood smear stained with Giemsa, showing a white
    blood cell (on left side) and several red blood
    cells, two of which are infected with Plasmodium
    falciparum (on right sideBlood smear stained with
    Giemsa, showing a white blood

6
  • - Field staining procedure. Field staining is a
    good method to stain thick films but is not
    suitable for thin films. However, it has the
    remarkable advantage to be extremely quick (the
    smear may be stained in 1 minute).
  • - Leishman staining procedure. Since Leishman
    staining solution uses methanol as a solvent,
    this method is only useful to stain thin films.

7
  • (B ) Quantitative Buffy Coat (QBC ) and the
    direct acridine orange staining
  • Is a sensitive microscopic test based on
    the ability of acridine orange to stain nucleic
    acid containing cells
  • A direct acridine orange (fluorochrome)
    staining This method recently proposed of thin
    and thick film to provide an economically
    convenient alternative to the QBC technique for
    use in the field by using specially designed
    interference filters that may be connected to
    conventional light (even sunlight) microscopes

8
  • (C) DNA probes and Polymerase Chain Reaction
  • The initial studies in nucleic acid-based
    malaria diagnosis used the parasites repetitive
    DNA found throughout the Plasmodium genome as the
    diagnostic target . Therefore, after the
    sequencing of two small subunits (18S) rRNA genes
    from P. falciparum and P. vivax ,
    species-specific regions of the rRNA genes have
    been exploited in developing a sensitive and
    specific

9
  • (D) Detection of P. falciparum antigen
  • The production of histidine rich protein
    II (HRP-II) antigen by blood stages of Plasmodium
    falciparum forms the basis for the development of
    ELISA antigen test and more recently, of the
    dipstick Becton Dickinson ParaSight -F test,
  • ( E) Other direct diagnostic methods
  • The determination of blood levels of
    parasite-specific lactate dehydrogenase (pLDH)
    has been evaluated as indirect method of
    quantifying parasitaemia and also drug resistance

10
  • 2- Indirect diagnosis (immunodiagnosis)
  • Detection of Plasmodia specific antibodies
    by
  • 1-Immunofluorescene (IFAT) The first serological
    test to be used for malaria antibodies was
    immunofluorescence (IFAT), which may give
    quantitative results for both G and M specific
    immunoglobulins. Its specificity and sensitivity
    largely rely on the laboratory technicians
    expertise.

11
2- Indirect haemoagglutination test (IHA) Is
simple and suitable for field studies, but its
sensitivity and specificity are poor 3-
Radioimmunoassay (RIA) is sometimes used but
needs well equipped research laboratories and
personnel.
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