Malarial parasite immunochromatogr

aphy technique

MADE BY SARA AZHARBS MEDICAL TECHNOLOGY

6TH SEMESTERLIAQUAT NATIONAL HOSPITAL

MALARIAItalian, Mala “bad” aria “air”

Malaria is a mosquito borne parasitic disease Caused by plasmodium parasites Transmitted by the sting of the Anopheles mosquito or by a

contaminated needle or transfusion Tropical and subtropical regions 300 to 500 million cases annually Mortality rate is 1.1-2.7 million / year One death every 20 to 30 seconds, somewhere in the world

HISTORY OF MALARIA

The first evidence of malaria parasites had been found in mosquitoes preserved in amber from the Paleogene period that are approximately 30 million years old

– 500 B.C_ Hippocrates Classified the fever types Noted relation ship b/w

enlarged spleen and marshes

– First Century AD_ Roman writersAttributed malarial diseases

to the swamps

– 1880-laveran_ discovered plasmodium in human blood

– 1885- Golgi_ erythrocytic schizogony in man

– 1894 – Manson_ role of mosquito in malaria

– 1857-Ronald Ross _ sexual cycle in mosquito

– 1948-Short and Graham _ pre erythrocytic schizogony – 1969 – Rudzinska – fine structure of plasmodium

Malarial parasite

There are four species of Plasmodium that cause Malaria in man :

P. falciparum P. vivax P. ovaleP. malariae

Lab Diagnosis

Method used to diagnose:

Microscopy Serology – Rapid Diagnostic Tests Molecular biology method (PCR)

Introduction of MPICT

Immunochromatographic test using monoclonal antibodies against the metabolic enzyme pLDH (parasite lactate dehydrogenase) of Plasmodium spp.

These monoclonal antibodies are classified into two groups:

One specific for Plasmodium falciparum And the other is a pan-specific monoclonal antibody

which can react with all four species of Plasmodium spp.

Sample material

Capillary blood collected from fingertip.

Whole blood collected by venipuncture, using EDTA sample tubes.

Positive control

Positive control is a recombinant pLDH giving exactly the same result as a blood sample from a patient infected with Plasmodium falciparum.

Sensitivity of test

MPICT can detect peripheral parasitaemia levels of 0.001-0.002% (50-100 parasite per micro litre of blood).

This sensitivity can be compared to microscopic observation of a thin blood smear using a 100x immersion objective.

Specificity

MPICT detects the presence of pLDH, an enzyme produced by both sexual and asexual form of the parasite.

There is no cross-reaction with human LDH or rheumatoid factor.

Test Procedure

Take a device, place it horizontally on a flat surface , write the name or number on the label.

Tear open the ampoule of buffer, add 1drop of buffer to the first well (conjugate well, marked with a red), and 4 drops to the second well (wash well). Allow to stand 1 minute.

Add the entire volume of blood by squeezing the pipette gently, to the first well .

Stir gently with the upper end of the pipette and allow to stand for 1minute .

Continued…

Hold the device with the wells between thumb and fore finger and, with the other hand, pull out the dipstick holder (with the label)

Place the wells back on the table, insert the legs of the dipstick holder into the holes beside the conjugate well so that the dipstick end reaches the bottom of the conjugate well.

Allow to stand for 10 min. The blood/conjugate mixture should then be completely

soaked up.

Continued…

Transfer the dipstick to the second well and allow to stand for 10 minutes. The reaction field should then be completely cleared of blood . The control band must be clearly visible.

Remove the dipstick from the wash well and click it back into the clear plastic piece.

Close the wells with the well cover, break them off, and break the two legs off from the clear plastic piece.

Discard them into a suitable waste container. Read the reaction and interprete the results.

Principle

In case of presence of Plasmodium in the blood sample, the pLDH captured by the conjugate reacts with the specific antibodies against Plasmodium falciparum and/or Plasmodium spp.

The reactions are demonstrated by the appearance of dark purple bands on the dipstick.

Goat anti-mouse used as procedure control.

Monoclonal anti-pLDH reacts with all 4 species of Plasmodium (P.falciparum/ P.vivax / P.malariae / P.ovale).

Monoclonal anti-pLDH specific only to P.falciparum.

Validation of the test

Results are valid if:

The control band is clearly visible.

The reaction field is cleared of blood.

Validation of the test

Results are not valid if:

The dipstick is not sufficiently cleared.( reaction field remains red).

The control band is not present.

The control band is not visible even if one or both of the diagnostic bands are present.

In such cases repeat the test.

Benefits

Fast answer : time-to-result never exceeding 20 minutes

Easy to use : just adding a drop of sample in the right container

Relatively inexpensive to make

limitations

MPICT detects the presence of pLDH from live parasite only. Comparison tests with PCR may therefore not be conclusive.

Any modification of the described test procedure or use of other reagents may modify the reaction pattern and invalidate the test.

Remember that the result of MPICT is to be interpreted within the epidemiological, clinical, and therapeutical context.

Thank youThank you