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 parasite’s nucleic acid or products in the blood Indirect Diagnosis: the demonstration of the patient’s 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) stainingThis 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 parasite’s repetitive DNA found throughout the Plasmodium genome as the diagnostic target . Therefore, after the sequencing of two small subunits (18S) rRNA genes from P. falciparumand 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 falciparumforms 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 technician’s 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.