Dr Piyush Tailor

MALARIA ANTIGEN, PLASMODIUM VIVAX & FALCIPARUM

Purpose of examination:
Four species of the plasmodium parasites are responsible for malaria infections in human viz. P.falciparum, P.vivax, P.ovale and P.malariae. Early detection and differentiation of malaria is important due to incidence of cerebral malaria and drug resistance associated with falciparum malaria and due to the morbidity associated with other malaria forms.
It is rapid, self- performing, qualitative, two site sandwich immunoassay, utilizing whole blood for the detection of P.falciparum specific histidine rich protein-2 and P.vivax specific pLDH.
The kit detects the presence of p.vivax specific pLDH For detectionof vivax malaria and P.falciparum specific histidine rich protein-2 for detection of falciparum malaria.

Principle of examination:
Utilizes the principle of agglutination of antibodies/antisera with respective antigen in immune-chromatography format along with use of nano gold particles as agglutination revealing agents. As the test sample flows through the membrane, assembly of the device after addition of the clearing buffer, the colored colloidal gold conjugates o0f the agglutinating sera for HRP-2 and agglutinating sera for pan malaria specific pLDH complexes the HRP-2/corresponding pLDH in the lysed sample. This complex moves further on the membrane of the test region where it is immobilized by the agglutinating sera for HRP-2and/or agglutinating sera for P. vivax specific pLDH antibody and/or agglutinating sera for pan malaria specific pLDH coated on the membrane leading to formation of a pink-purple colored band in the respective regions which confirms a positive test result. Absence of a colored band in the test region indicates a negative test result for the corresponding antigen. The unreacted conjugate along with the rabbit globulin-colloidal gold conjugate and unbound complex if any, move further on the membrane and are subsequently immobilized by the agglutinating sera for rabbit globulin coated on them, membrane at the control region, forming a pink-purple band. The control band formation is based on the ‘rabbit/agglutinating sera for rabbit globulin’ system. Since it is completely independent of the analyte detection system, it facilitates formation of consistent control band signal independent of the analyte concentration. This control band serves to validate the test performance.

Performance specification:
Sensitivity: 100% to P. vivax and P. falciparum

Sample type required:
Anticoagulated whole blood (EDTA).
Stored at 2’C-8’C after analysis for 24 hrs.

Preservative needed:
Sample collected in EDTA bulb

Reagents required:
1. Test cards (Devices)
2. Pipette: Plastic sample Applicator
3. Clearing buffer in the dropper bottle

Calibration method:
Not applicable

Detailed work bench instruction:
PROCEDURE :

In accordance with good laboratory practice, all calibrators, controls and samples should be considered potentially infectious and should be handled with care. The personnel mandatorily should use protective laboratory coats, gloves and eye glasses.
• Remove the test card from the pouch just prior to use and bring it at room temperature.
• Lay the card flat on the work surface
• Add 5uL of sample slowly using the pipette on to the box labeled “A”
• Add 2 drops of clearing buffer on to the box labeled “B”
• Read the result after 20 min.
Precautions:
• Optimal result will be obtained by adherence of this protocol.
• Do not reuse test card.
• Biological contamination of dispensing pipette can lead to false positivity.
• Do not use cards beyond expiry date.
• Do not mix reagents in different lots.
• Dispense accurate amount of sample

Troubleshooting:

Most blood samples clear within test running. However in few fresh samples & stored samples, the background clearance maybe delayed for 15 to 20 mins more. In such cases if the background of the test window has not cleared within 15 mins., place a drop of clearing buffer. Wait for another 15 mins before reading the result.

Quality control procedure:
1. The test cards have an internal positive control band labeled as “C”. This band must show positivity with each run.
2. weekly minimum one malaria smear is cross checked with test card for malaria antigen and results are recorded in C\Records\File\9\Internal Quality Control Records

Interference:
• Any alteration of the test procedure will make the test invalid
• Interference due to presence of heterophile antibodies in patient’s sample can lead to erroneous analyte detection in immunoassay

Calculation of results and uncertainty:
• Cannot identify carriers due to absence of HRP-II in gametocyte
• In endemic areas of high prevalence of mixed infection, need for speciation under microscopy for correct therapy

Biological reference interval:
Negative : Only one pink-purple band appears at control region “C”

Reportable interval for examination results:
Negative : Only one pink-purple band appears at control region “C”
Positive : For P.Falci.- In addition to control band, two pink colored bands appear at region ‘Pf’ and ‘Pan’ respectively in the test window.
For P. Vivax- In addition to control band, one pink colored bands appear at region ‘Pv’ and ‘Pan’ respectively in the test window.
For other species – In addition to contr4ol band, one pink-purple band appears only at “Pan” region.
Positive for mixed infection – In addition to control band, three pink-purple bands appear at regions ‘Pf’, ‘Pv’ and ‘Pan’ respectively.
Invalid result – the test should be considered invalid if no bands appear on the device. The test should also be considered invalid if only test bands (Pan and/or Pv and/or Pf) appear and no control band appears. Repeat the test with a new device ensuring that the test procedure has been followed accurately.
Critical values:
Positive case of p.falciparum malaria; if smear shows +4 positive

Interpretation by the laboratory:

• Test result must be correlated with the clinical findings
• Interpret test result in context with epidemiological, clinical & therapeutical findings
• In P. falciparum malaria infection, PF HRP-2 is not secreted in gametogony stage. Hence, in carriers the PF band may be absent.
• Since PF HRP-2 persists for upto a fortnight even after successful therapy, a positive test result does not indicate a failure of therapy. If reaction of the test result remains positive with the same intensity after 5 to 10 days, post-treatment, the possibility of a resistant strain of malaria has to be considered.
• P. vivax band can be used for monitoring success of antimalarial therapy in case of stand alone P. vivax infection. For monitoring success of antimalarial therapy in case of stand alone P. falciparum infection or mixed infection, employing a panspecific pLDH based system is recommended after 5 to 10 days of initiation of the therapy.
• In case of infection due to P. Vivax are falci or due rto mixed infections by these species, the pan malaria band will also be positive. Hence, differentiation of infection du8e to P.Ovalae or P.malarie can not be done.
• In a cases, where thw HRP-2 band is positive and the ‘Pan’ malaria band is negative, it may indicate a case of post treatment malaria/untreated malaria. Retesting after 2 days is advised, in such cases.
• Potential sources of variability:
1. Lipemic
2. Hemolysed
3. Clotted
4. Broken vial
5. Identification error
Reference:
1. Test insert of Paramax-3
2. Robbins Pathologic basis of disease, 8th edition,2010