This document discusses various techniques for diagnosing malaria, including microscopy, immunological techniques, and molecular techniques. Microscopy techniques include thick and thin blood smears, which are the gold standard, and the quantative buffy coat test. Immunological techniques include indirect fluorescent antibody testing, enzyme-linked immunosorbent assay, and rapid diagnostic tests targeting antigens like HRP-2, plasmodium aldolase, and lactate dehydrogonase. Molecular techniques include polymerase chain reaction and other nucleic acid amplification techniques, which can detect malaria parasites with greater sensitivity than microscopy.

1. Dr. Kainat Panjwani, PharmD
Asst. Professor
Pharmacotherapeutics II
MLR Institute of Pharmacy

2. Laboratory diagnosis of malaria requires the
identification of the parasite or its antigens/ products in
the patient’s blood.
The requirements of a diagnostic test are specificity,
sensitivity, ease of performance and a reasonable cost.
Current available techniques can be separated in three
categories:
1. Microscopy
2. Immunological techniques
3. Molecular techniques

3. a) Thick and thin blood smear study
Thick and thin blood smear study is the gold
standard method for malaria diagnosis. The procedure
follows these steps: collection of peripheral blood,
staining of smear with Giemsa stain and examination
of red blood cells for malaria parasites under the
microscope.

4.  Thick smear. It is not fixed in methanol; this allows the
red blood cells to be hemolyzed, and leukocytes and any
malaria parasites present will be the only detectable
elements. However, the hemolysis may lead to distorted
plasmodial morphology making plasmodium species
differentiation difficult. Therefore, thick smears are mainly
used to detect infection and to estimate parasitemia.
 Thin smear. It is fixed in methanol. Thin smears allow the
examiner to identify malaria species, quantify parasitemia,
and recognize parasite forms like schizonts and
gametocytes.

5. SCHIZONTS TROPHOZOITES GAMETOCYTES SCHIZONTS

6. b) Quantative Buffy Coat (QBC) test
This method involves centrifuged
and compressed red blood cell layer
stained with acridine orange and
then examinated under an ultra-
violet light source. The whole
procedure takes place in a glass
hematocrit tube which is precoated
internally with acridine orange stain
and potassium oxalate; it is filled
with 55-65 μl of blood. The tube is
centrifuged and so the components
separate according to their densities
forming bands.

7. Fluorescing parasites are then observed, with a UV
microscope, at the red blood cell/white blood cell interface
as shown below:
QBC test is easier and faster than classic peripheral blood
smear microscopy but the equipment required is expensive
and species identification and accurate enumeration are
impossible.

8.  Antibody-based techniques
a) Indirect fluorescent antibody test (IFAT)
The antigen consists of infected blood bound to a 12-spot microscope
slide. A drop of diluted washed infected red blood cells is placed on
each spot and allowed to dry. It is then incubated with the serial
dilutions of the test serum, followed by a solution of anti-human
immunoglobulin labeled with fluorescein isothiocyanine which
contains Evans blue as a counterstain. When the slides are dried, they
are examined by fluorescence microscopy.
Antibody in the test serum reacts with antigen of parasites and the
anti-immunoglobulin reaction with the antibody is demonstrated by
the fluorescence of the parasites as shown

9. b) Enzyme- linked immunosorbent assay (ELISA)
This method uses a soluble malarial antigen coated on the walls
of a microtitre plate,
If the test is positive, the antibody binds the antigen resulting in a
visible colour change.
When the test is negative, in the absence of antibody, there is no
change of colour of the substrate.

10.  Antigen-based techniques
Rapid Diagnostic Test (RDT)
RDT is a device that can detect malaria antigen in a small amount of
blood (5μl) by immunochromatographic assay (colour change in an
absorbing nitrocellulose strip) with monoclonal antibodies directed
against the parasite antigen. Depending on the target antigen, rapid
tests that now exist may involve combinations of the following:
 HRP-2 (Histidine Rich Protein-2) is a protein produced by the
asexual stages and gametocytes of P. falciparum, expressed on the
membrane of red blood cells (sensitivity: detects parasitemia of >40
parasites/ μl). It often persists in patient’s blood for weeks after
successful treatment.
 Plasmodium aldolase is an enzyme of the parasite’s glycolytic
pathway expressed by all malaria species(pan malarial antigen- PMA).
 Lactate dehydrogonase (LDH) is a glycolytic enzyme produced by
asexual and sexual stages of parasites (PF, PV) and released by infected
red blood cells. (sensitivity: detects parasitemia of >100 parasites/ μl)

11. The PfHRP2 test strips have 2 lines, one for the
control and the other for the PfHRP2 antigen.
The PfHRP2/PMA test strips and the pLDH
(parasite LDH) test strips have 3 lines, 1 for control,
and the other 2 for P. falciparum and non-
falciparum antigens.
Change of color on the control line is necessary for
the test to be validated. With color change only on
the control line and not in the other lines, the test is
regarded as negative.
In PfHRP2 test, color change on both the lines is
interpreted as a positive test for P.
falciparum malaria (Figure 7).
With the PfHRP2/PMA and the pLDH tests, color
change on the control line and the pan specific line
indicates non-fa1ciparum infection and color change

12.  Polymerase Chain Reaction (PCR)
Using PCR amplification, it is possible to detect all 4
species of malaria parasites with a reportedly 10-fold
greater sensitivity than microscopy. New technologies
such as saponin lysed erythrocytes NAT (nucleic acid
amplification technique) and LAMP (loop- mediated
isothermal amplification) can provide a lower-cost
diagnosis with greater sensitivity and specificity for the 5
plasmodium species accepted by WHO.