OptiMAL Test for Rapid Diagnosis of Malarial Parasites: Introduction, Principle, Procedure, Result Interpretation, Clinical Significance and Keynotes

OptiMAL Test for Rapid Diagnosis of Malarial Parasites: Introduction, Principle, Procedure, Result Interpretation, Clinical Significance and Keynotes

Introduction of OptiMAL Test

OptiMAL Test is an immuno-chromatographic assay, using monoclonal antibodies against the metabolic enzyme parasite lactate dehydrogenase (pLDH ) of Plasmodium species. Plasmodium is the causative agent of infectious disease, malaria. These monoclonal antibodies are classified into two groups:  one specific for  P. falciparum and the other is a pan-specific monoclonal antibody that reacts with all four species of Plasmodium species which can occur in human beings: P. falciparum, P. vivax, P. ovale, and P. malariae. The test is simple and rapid assays can be completed in 15-20 minutes. It reduces the need for trained examiners and costly equipment. The test kit is sortable at room temperature.

Principle of OptiMAL Test

OptiMAL Test works under the principle of immunochromatography and the immunochromatographic test principle is the same as the sandwich ELISA method. The only difference is that immunological reaction is carried out on the chromatographic paper by capillary action. For this system, two kinds of specific antibodies against the antigen are used. One of the antibodies is immobilized on the chromatographic paper while another is labeled with colloidal gold and infiltrated into a sample pad. An immunochromatographic unit is completed by attaching the sample pad at the end of the membrane. The liquid sample is dropped on the sample pad. The antigen in the sample forms an immunocomplex with the antibody labeled with colloidal gold. Its complex moves along with the liquid sample and makes a contact with the antibody immobilized on the membrane. It is followed by forming an immuno- complex with the immobilized antibody. It results in generating a colored red-purple line.  The appearance of a red-purple line on the membrane indicates the presence of antigen of interest in the sample. The liquid of the sample migrates through the membrane very fast, it makes it possible to detect the presence or absence of antigen within 15 minutes. In the case of the presence of Plasmodium spp. in the blood sample, the pLDH captured by the conjugate reacts with the specific antibodies against Plasmodium falciparum and/or Plasmodium spp.

Test Requirements for OptiMAL Test

Kit (Bio-Rad) provides-

  1. a device with dipstick, conjugate well, wash well
  2. well cover
  3. dropper ampoule with buffer
  4. lancet
  5. disinfecting swab
  6. Disposable plastic pipette (printed mark for 10 µL)
  7. schematic test procedure/manual/leaflet

Extra we need-

  1. Gloves
  2. Waste bin
  3. Marker
  4. Blood: Capillary blood collected from the fingertip or Whole blood collected by venipuncture, using EDTA sample tubes

Procedure of OptiMAL Test

(Bio-Rad-Device Method)

  • Tear and open the aluminum package and take out all the material.
  • Take the device, place it horizontally on a flat surface, write the patient’s ID or number on the label.
  • Tear open the ampoule of buffer, add a  drop of buffer to the first well (conjugate well, marked with a colored line), and four drops to the second well (wash well). Allow standing for one minute.
  • For finger-prick blood: Clean the fingertip with the swab, let dry, remove the lancet from its envelope, prick the finger. Take the pipette, squeeze it, place the open tip into the blood drop, release pressure and draw up blood to the black line. Discard used swab and lancet into a suitable waste container. When using venous blood, draw blood from the tube into the pipette in the same manner.
  • Add the entire volume of blood by squeezing the pipette gently, to the first well (conjugate well, marked with a colored line).
  • Stir gently with the upper end of the pipette and allow to stand for one minute. Discard the pipette into a suitable waste container.
  • Pull the optimal test device apart: hold the device with the wells between thumb and forefinger 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 (with colored line) so that the dipstick end reaches the bottom of the conjugate well. Allow standing for ten minutes. The blood/conjugate mixture should then be completely soaked up.
  • Transfer the dipstick to the second well (wash well) and allow it to stand for ten 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 interpret the results.

Result Interpretation of OptiMAL Test

  • Test Negative: Only one band at the control region
  • Test Positive: Two or three bands one at the control region while another at the test region/s: Test Positive.
  • Positive for P. vivax, or P. malariae, or P. ovale: Bands at the control and mix region while no band at P. falciparum regionPositive for P. falciparum: Bands at all three regions (as shown above image)
  • Test Invalid: The control line fails to appear. Insufficient specimen volume or incorrect procedural techniques are the likeliest reasons for control line failure. Repeat the test using a new test device

Limitation of OptiMAL Test

  1. OptiMAL Test detects the presence of pLDH from live parasites only and thus the possibility to monitor the efficiency of treatment. Comparison of this test with PCR may therefore not be conclusive.
  2. Any modification of the described test procedure or use of other reagents may modify the reaction pattern and therefore test will invalidate.
  3. Remember that the result of this test is to be interpreted within the epidemiological, clinical, and therapeutical context.

Clinical Significance of OptiMAL Test

The optiMAL test is only positive when live parasites are present in the blood. A repeat test becomes negative generally within 2-4 days following the beginning of successful treatment.  Thus this optimal test is suitable to verify the effectiveness of therapy, underlining possible resistant strains of Plasmodium species.

Keynotes on OptiMAL Test,

  • OptiMAL Test detects the presence of pLDH, an enzyme produced by both sexual and asexual forms of the parasite. There is no cross-reaction with human LDH or Rheumatoid factor. The “lysis and dilution” buffer saves also non-specific reactions due to heterophile antibodies.
  • This OptiMAL test detects 50-100 parasites per µL of blood.
  • This OptiMAL test is fast  (20 minutes) and easy to perform even the test kit provides all the necessary materials to perform a test.
  • BinaxNOW® Malaria Test is the only available RDT for malaria in the United States.
  • Be aware while handling patient samples and any material that comes directly in contact with them as potentially able to transmit infectious diseases.

Further Readings on OptiMAL Test

 

  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC124834/
  • https://commerce.bio-rad.com/webroot/web/pdf/inserts/CDG/en/710024_881177_EN.pdf
  • https://www.lagaay.com/shop/product/14341
  • https://www.researchgate.net/publication/344678889_Optimal_test-
  • https://www.cdc.gov/dpdx/diagnosticprocedures/blood/antigendetection.html
  • https://apps.who.int/iris/bitstream/10665/204118/1/9789241510035_eng.pdf%3Fua%3D1
  • https://www.sciencedirect.com/topics/medicine-and-dentistry/malaria-rapid-test

 

 

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