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Introduction of Leptospira Rapid Diagnostic Test 

Leptospira rapid diagnostic test is for both present and past infection status, immunoglobulin M (IgM) indicates present history whereas immunoglobulin G (IgG) indicates past as shown above picture. Advantage Lepto IgG/IgM Combo Rapid Test is a visual, rapid, sensitive, qualitative immunoassay for the detection of Leptospira-specific IgM and IgG antibodies in human serum or plasma or whole blood ( CTK, SD BIOSENSOR, Diagnostic Automation Kits). Leptospirosis is an infectious disease. It is a worldwide occurring zoonotic disease and its causative agent is pathogenic spirochetes of the genus Leptospira and species L. interrogans.  Mode of infection: Although traditionally considered as an occupational risk among persons exposed to contaminated water or infected animal urine, leptospirosis is becoming recognized as a common cause of febrile illness in tropical environments worldwide. Pathogenesis of leptospirosis: Bacterium enters the human body through cuts or abrasions on the skin or through intact mucosa of the mouth, nose, or conjunctiva. Clinical features: from a mild catarrh like fever, chills, nausea, muscle aches to icteric disease such as Weil’s syndrome, which are characterized by renal failure, liver impairments, and hemorrhages and have a high mortality rate. Sign and Symptoms:  Clinical signs and symptoms of this infection resemble those of many other infectious diseases including Viral hemorrhagic fever and Dengue fever, clinical findings need to be confirmed by laboratory diagnostic techniques.  This Lepto IgG/IgM Combo Rapid Test is intended to be used as a screening test by professionals and provides a preliminary test result to aid in the diagnosis of infection with Leptospira interrogans. Any interpretation or use of this test result must also rely on other clinical findings as well as on the professional judgment of health care providers

Principle of the Test

Lepto IgG/IgG Combo test is based on the immunochromatographic assay. The test uses monoclonal anti-human IgM antibody (test line M) and monoclonal anti-human IgG (test line G) immobilized on a nitrocellulose strip. The test sample is introduced to and flows laterally through an absorbent pad where it mixes with the conjugate. The conjugate contains colloidal gold conjugated to a Leptospira antigen which is prepared from the culture of bacteria. If the sample contains Leptospira-specific IgM and/or IgG antibodies, it forms a complex with the Leptospira antigen gold conjugate making antigen antibodies complex. This complex then migrates through the nitrocellulose membrane by capillary action. When the complex meets the line of immobilized monoclonal antibodies ( ‘M & G’), it generates a red/pink line, indicating that the sample is reactive. A red/pink procedural control line should always develop at the ‘C’ region to indicate that the test has been performed properly ( as internal quality control).

Sensitivity and specificity of different kits for  Leptospira rapid diagnostic test

1. Jmitra: Sensitivity : 98.16% (for IgM Antibodies) and 100% (for IgG Antibodies)
   Specificity: 99.61%
2. CTK Biotech, Inc.:The OnSite Leptospira IgG/IgM Combo Rapid Test shows 100% specificity and 100% sensitivity
3. Diagnostic Automation/Cortez Diagnostics, Inc. : For IgM test -sensitivity: 90%
   specificity: 99.0% and IgG- sensitivity: ＞99.9% and  specificity: 99.0%

Requirements for Leptospira Rapid Diagnostic Test

1. Test kit and it contains-Individually sealed foil pouches containing:
   a. One cassette device
   b. One desiccant
   2. 5 μL capillary tubes
   3. Sample diluent
   4. Instructions for Use
2. Test specimen( whole blood/serum/plasma)
3. Marker
4. Timer
5. Waste bin
6. 0.5% Bleach (Sodium hypochlorite)

Test Procedure 

1.  Bring the specimen and the test components to room temperature if refrigerated or frozen. Once thawed, mix the specimen well prior to performing the assay.
2.  Place the device on a clean, flat surface.
3. Be sure to label the device with the specimen’s ID number.
4. Fill the 5 µL capillary tube with the specimen not to exceed the specimen line. The volume of the specimen is approximately 5 µL. For maximum precision,
   transfer the specimen using a pipette capable of delivering a volume of 5 µL.
   Holding the capillary tube vertically, dispense the entire specimen into the center of the sample well (Swell) making sure that there are no air bubbles.
   Immediately add 2 drops (approximately 60-80 µL) of sample diluent into the buffer well (B well) with the bottle positioned vertically.
5. Set up a timer.
6. Results can be read at 15 minutes. Positive results can be visible in as soon as 1 minute.
7. Negative results must be confirmed at the end of the 20 minutes only. Any results interpreted outside of the 15-20 minute window should be considered invalid and must be repeated.
8. Discard used devices after interpreting the result following local requirements governing the disposal of devices.

Result Interpretation

IgG test positive:  Two distinct colored lines appear. One color line should be in the control region (C) and another color line should be in the IgG region.
IgM test positive: Two distinct colored lines appear. One color line should be in the control region (C) and another color line should be in the IgM region.
IgG and IgM test positive: Three distinct colored lines appear. One color line should be in the control region (C) and another two color lines should be in the IgG and IgM region.

Test negative: One color line appears in the control region (C). No apparent red or pink line appears in the IgG and IgM region.
Test invalid: Control line fails to appear. Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control line failure. Review the procedure and repeat the test with a new test cassette. If the problem persists, discontinue using the test kit immediately and contact your local distributor.

Keynotes

• Do not reuse test cards.
• Do not use the kit beyond the stated expiry date mentioned on the kit.
• Interpret the results at the end of 20 minutes only. Do not read results after 20 minutes as it may give erratic results.
• Use a separate sample dropper for each sample and discard it as
  bio-hazardous waste.
• The intensity of the color in the test line region (T) will vary depending on the concentration of Leptospira antibodies present in the specimen. Therefore, any shade of red in the test region should be considered positive.

Limitation of the test

• The advantage Lepto  IgG/IgG Combo test is for in vitro diagnostic use only.
• The test is a qualitative screening assay and is not suggested for use in determining quantitative levels.
• This is only a screening test. All samples detected reactive must be confirmed by using a confirmatory test such as the Microscopic Agglutination Test (MAT).

References

1. https://ctkbiotech.com/product/leptospira-igg-igm-combo-rapid-test-ce/
2. http://jmitra.co.in/download/Procedure/Manual-AdLeptospiraIgMIgGCard.pdf
3. https://www.rapidtest.com/pdf/Leptospira%20IgG-IgM%20(2016-06-21).pdf
4. http://sdbiosensor.com/xe/product/4096?category=2306
5. https://athenesedx.com/insert/R0101C.pdf