Laboratory diagnosis of Malaria

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Laboratory diagnosis of Malaria
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• 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).

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• (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.

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• - 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

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• 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

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• - 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.

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• (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

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• (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

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• (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
  

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• 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.

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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.