Experiment No.

14
SEROLOGIC DIAGNOSIS OF PARASITIC INFECTIONS

A. Rapid Diagnostic Test for Plasmodium SP.

5 INTRODUCTION
Malaria is a protozoan disease caused by Plasmodium sp. That is transmitted by female
Anopheles mosquitoes. Its management requires prompt diagnosis before effective treatment is
administered. The necessitates the use of rapid diagnostic test (RDTs) thet are relatively easy to
perform and provide results that are comparable to the gold standard: microscopic examination of
10 thick and thin blood smear. Malaria RDTs usually detect the following antigens: histidine-rich
proteins 2 (HRP2) for P. falciparum and P-lactate dehydrogenase (LDH) for P. vivax and other
species. These immunochromatographic tests may detect either single or multiple infections.

PRINCIPLE
15 Rapid diagnostic test for the detection of malarial antigens are based on
immunochromatography. It involves antigen capture using monoclonal antibodies conjugated to
gold particles in mobile phase. The antigen-antibody complex form is captured an immobilized
secondary monoclonal antibody to produce a visible colored line.

20 SPECIMEN
Human serum or plasma

PROCEDURE
1. Prepare the test kits. Let it stands at room temperature prior to testing.
25 2. Dispense a drop of blood on the sample hole of the cassette.
3. Dispense three drops of lysing agent in the buffer hole. The buffer carries the blood along
the length of the RDT.
4. Allow the test to stand for 20 minutes.
5. Observe for the formation of test band.
30
MANNER OF REPORTINF RESULTS
Positive- distinct colored band on the both test and control lines
Negative- distinct colored band on control line only
Invalid- control band fail to appear on the result window
35
Discussion notes:
Rapid diagnostic tests for malarial parasite diagnosi are based on immunochromatographic
 detection of antigen-dye-labelled antibody complex. Initially malarial parasite proteins react with
specific antibodies tagged with certain dye. The complex is captured on the strip by the band of
the bound antibody, forming a colored visible line on the T-test line of the result window.

5 Current approaches in RDTs target the following three relevant antigens:

1. Histidine-rich Protien 2 (HRP-2), specific to plasmodium falciparum.
 Heat stable water-soluble protein produces by asexual forms and young
gametocytes of P. falcifarum.
 First antigen to be employed in development of RDT.
10  It is expressed on the membrane surface of infected red cells.

2. Parasite-specific Plasmodium lactate dehydrogenase (pLDH)

 This is an enzyme seen on the glycolytic pathway of Plasmodium sp. that
generated both by sexual and asexual forms of parasite.
15  Several isomers of LDH are observed for different species of Plasmodium, thus it
may be either specific for one species or pan-specific for all.

3. Aldolase
 An enzyme of glycolytic pathway of mlarial parasite
20  Suggested as target antigens for non-P. falciparum

Summary table of three malarial antigens and their target Plasmodium sp

Target Malarial Species HRP2 PLDH Aldolase

P. Falciparum specific √ √
25 Pan-specific (all species) √ √
P. vivax specific √
B. TOXOPLASMA IGG/IGM RAPID TEST
Toxoplasmosis is a parasitic disease caused by a tissue coccidian called Toxoplasma
gondii. Members of the Felidae family are definitive host of this protozoan. Humans accidentally
acquire the infection through any of the any of the following routes: ingestion or drinking of
contaminated food and water containing sporulated oocysts; consumption of undercooked or raw
30 meat containing tissue cysts; or via blood transfusion, organ transplantation, and vertical
transmission. Immunocompetent persons are asymptomatic and develop immunity to the
disease. On other hand, immunocompromised patients may develop chorioretinitis, encephalitis,
and focal brain lesions. Pregnant woamen are also at risk because T. gondii can cross the
placenta and cause severe congenital defects. IgM rapid test detects IgG antibodies against
35 Toxoplasma that can aid in the diagnosis of the disease.
 PRINCIPLE
Toxoplasma IgG/IgM is a rapid test that utilizes a solid phase immunochromotographic
technique. This qualitative test differentially detects IgM and IgG antibodies against T. gondii in
5 the serum/plasma.
In the Toxoplasma IgG/IgM assay, the specimenis added to the sample port and flows
through the absorbent pad. If IgG/IgM anti-Toxoplasma antibodies are present, these combine
with recombinant Toxoplasma-colloidal gold conjugate to form an immune complex.
The reaction port has a nitrocellulose membrane strip with two lines, the G line and M line,
10 which are pre-coated with mouse monoclonal anti-human IgG and mouse monoclonal anti-human
IgM anti-bodies, respectively. As the complex of IgG/IgM anti-Toxoplasma antibodies-dye
conjugate diffuse through this reaction area, it will be captured by the respective monoclonal anti-
human IgG and anti-human IgM antibodies immobilized in the test lines and forms a red-colored
line. Excess reagents or unreacted components will bind with the goat anti-mouse IgG antibody
15 immobilized in the control line, forming another red-colored line.

SPICEMEN
Human serum or plasma

20 PROCEDURE
1. Allow the specimen and test components to reach room temperature.
2. Remove the test device from the pouch and place it on a falt surface.
3. Use micropipette or disposable plastic dropper to deliver 15 uL serum/plasma sample to the
sample well marked as “S”.
25 4. Allow 30 seconds to elapse before delivering three drops of assay dilluent onto the sample
well. This allows the serum/plasma to absorbed completely by the sample well.
5. Interpret the results after 15-20 minutes. Do not read the results beyond 20 minutes.

30

35 INTERPRETATION OF THE RESULT

Band Lines Results Interpretations

 Observed
C M G
+ - - Negative Absence of antibodies against Toxoplasma gondii
+ + - Positive Presence of IgM antibodies against T. gondii
+ - + Positive Presence of IgG antiboides against T. gondii
+ + + Positive Presence of both IgG/IgM antibodies against T. gondii
The control line fails to appear. Insufficient specimen volume
- - - Invalid or incorrect procedural techniques are most likely the reasons
for the control line failure. Repeat the test using a new test
technique.
Note:
[+]: presence of colored band on specific area
[-]: absence of colored band on the specific area