Diagnosis

A) Clinical Diagnosis

• Confusion, coma, neurological focal signs, severe anemia, and respiratory difficulties are more striking and may increase the suspicion index for malaria.
• Is usually sufficient to warrant treatment.
• Not typical and need to be confirmed by a laboratory test.

B) Microscopic Diagnosis

• Detection of parasites(P. falciparum) in stained blood films.
• This method used to differentiate between different parasite species and stages of the life cycle.
• The specimen is stained with the Giemsa stain to give to the parasites a distinctive appearance.
• Thick blood films are used in routine diagnosis and as few as one parasite per 200 μl bloods can be detected.
• In this additional laboratory findings may include mild anemia, mild decrease in blood platelets (thrombocytopenia), elevation of bilirubin, aminotransferases, albuminuria, and the presence of abnormal bodies in the urine.
• From the microscopic diagnosis, we can diagnose that the person is infected with malaria and the Blood Stage Parasites in thin blood smears and thick blood smears. Here are some images about the Stages of P. falciparum found in blood.

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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 P. falciparum (on right side).
  

C) Rapid Diagnostic Test:

The picture above demonstrates a positive test for P.falciparum.

• An alternate way of quickly establishing the diagnosis of malaria infection by detecting specific malaria antigens in a person's blood.
  
• It is recommended that all RDTs are followed-up with microscopy to confirm the results and to quantify the proportion of red blood cells that are infected.
  

Advantages:

※ Decrease the amount of time that it takes to determine that a patient is infected with malaria

Disadvantages:

※ May not be able to detect some infections with lower numbers of malaria parasites circulating in the patient’s bloodstream.

※ The currently approved DT detects 2 different malaria antigens; one is specific
for P. falciparum and the other is found in all 4 human species of malaria.

※ Thus, microscop is needed to determine the species of malaria .


D) Serology :

• Detects antibodies against malaria parasites using either indirect
  immunofluorescence (IFA) or enzyme-linked immunosorbent assay (ELISA).
  
• Used to assess past malaria experience but not current infection by malaria parasites.
  

Indirect immunofluorescence (IFA):

-> Used to determine if a patient has been infected with Plasmodium via the time required for development of antibody and also the persistence of antibodies.

-> Blood stage Plasmodium species schizonts (meronts) are used as antigen. The patient's
serum is exposed to the organisms; homologous antibody.

-> It will attaches to the antigen, forming an antigen-antibody complex if it present.

-> Fluorescein- labeled anti-human antibody is then added, which attaches to the patient's malaria-specific antibodies.

-> When examined with a fluorescence microscope, a positive reaction
is when the parasites fluoresce an apple green color.

The fluorescence indicates that the patient serum being tested contains antibodies that are reacting with the antigen preparation (P.falciparum).

Enzyme-linked immunosorbent assay:

-> Screen blood donors, but have limited sensitivity due to use of only Plasmodium
falciparium antigen instead of antigens of all four human species.

E) Molecular Diagnosis

• Detection of parasite genetic material through polymerase-chain reaction (PCR)
  techniques.
• Detection and speciation of Plasmodium is done with a two step nested PCR using the primers of Snounou et al 1993. Specific primers have been developed for each of the four species of human malaria.

The steps are:
~ In the first step (PCR1), 1 µl of extracted DNA is amplified using genus specific primers.1 µl of PCR1 amplification product is further amplified using primers specific.

~ Ten microliters of each PCR2 amplified DNA product is electrophoretically resolved on a 2% agarose gel then stained for 15 min with ethidium bromide and visualized by UV illumination for analysis of result.

Disadvantages:

※ High cost, high degree of training required.

※ Need for special equipment, absolute requirement for electricity

※ Potential for cross-contamination between samples.