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 Introduction and Importance of rK39 Test in Kala-zar

rK39 test result for visceral leishmaniasis (VL) as shown above image. Visceral leishmaniasis or Kala-azar is an intracellular protozoal infection caused by Leishmania donovani and transmitted by phlebotomine sandflies. VL is a severe disease with high mortality, endemic in 88 countries including 17 developed nations. A serious problem in much of the world including Brazil, China, East Africa, India, and areas of the Middle East, leishmaniasis is also endemic in the Mediterranean region including southern France, Italy, Greece, Spain, Portugal, and Northern Africa. In areas where leishmaniasis is endemic, recent migration patterns of people, vectors (sandflies), and reservoirs (dogs) have led to the urbanization of VL. In Southern Europe, VL has become the leading opportunistic infection in AIDS patients. VL is caused by members of the Leishmania donovani complex and canines have been identified as the major reservoir for transmission.

A rapid dipstick test based on the recombinant K39 protein is available for rapid diagnosis of kala-azar. K39 is an epitope apparently conserved on amastigotes of Leishmania species that cause visceral infection. Using K39 antigen-impregnated nitrocellulose strips developed for field conditions, fingerstick-obtained blood and serum samples tested from Indian subjects demonstrated a positive anti-K39 immunochromatographic reaction in 362 patients with aspirate-proven kala-azar; with an estimated sensitivity of 100% and a specificity of 97%. The strip testing proved simple to perform and yielded results within five minutes.

Importance of rK39 Test

Diagnosis and treatment of Kala-azar are problematic because of a variety
of reasons. While treatment is lengthy and relatively costly, definitive diagnosis of Kala-azar requires tissue specimens, which are conventionally obtained by organ needle aspiration for microscopic demonstration of amastigote forms in stained smears. Bone marrow and the spleen and, in some regions, lymph node are the tissues most often sampled in patients with suspected infection. The diagnostic sensitivity of splenic aspiration is high (95% – 98%), but the procedure carries a risk of bleeding; the sensitivity of examination of bone marrow specimens is considered to be lower (53% – 95%). Organ aspiration and accurate examination of smears also require technical skills that are not uniformly available everywhere. Culture or PCR testing of aspirate material improves parasitologic yield, but these methods are seldom undertaken outside of research laboratories. In the Kala-azar endemic areas of India, Napierís aldehyde test has been used for a long time. The test relies on the jellification caused by the binding of the serum globulins to the formaldehyde. The serum globulins increase in a variety of infections and thus this test is rather non-specific. A positive reaction may also be seen in diseases like Tuberculosis, Cirrhosis of the liver, Malaria, etc. Further, in kala-azar, the test becomes positive only when the infection is at least three months old and may remain positive even after six months of cure. Therefore a rapid, accurate, field suitable, and non-invasive method of diagnosis of kala-azar has long been sought to circumvent the need for obtaining tissue specimens and preceding limitations. A range of assays has been developed to detect antileishmanial antibodies. The complement fixation test detects specific antibodies present in the serum. The test though more sensitive as compared to the aldehyde test shows cross-reaction in cases of pulmonary tuberculosis, leptomonas leprosy, and Mycobacterium infections. Immuno-fluorescent antibody test (IFAT) in which parasite antigen labeled with a fluorescent dye is conjugated with serum antibodies and seen under the fluorescent microscope has also been widely used. The indirect hemagglutination test (IHA) is also based on the principle of antigen-antibody reaction. The serum antibodies are conjugated with parasite antigens to observe agglutination. Enzyme-Linked Immunosorbent Assay (ELISA) utilizes soluble antigen or sonicated extract of promastigotes to capture antibodies specific to Leishmania. Though sensitive and specific, it may give cross-reactions with infections like malaria, tuberculosis, leprosy, etc. at very low liters. Direct Agglutination Test (DAT) another test widely used for serodiagnosis of kala-azar is based on antigen-antibody reaction. Trypsin treated, stained, and formalin preserved promastigotes are used as antigens which shows agglutination with specific antibodies present in patients’ serum. The test is performed at room temperature through the antigens are stored under controlled temperature in the freezer. The usefulness of the above-mentioned serological tests is limited by, their variable sensitivity or specificity, the requirement of electricity, refrigeration, or a well-equipped laboratory, and high cost. Recently developed rapid dip-stick test rK39 is the option available now to diagnose kala-azar cases at the grassroots in conjunction with the clinical diagnosis.

Principle of rK39 Test

The kala-azar dipstick rapid test is an immunochromatographic assay for the qualitative detection of antibodies to L. donovani in human serum. The assay is for aid in the presumptive diagnosis of VL. During testing the serum sample reacts with the dye conjugate (Protein A- Colloidal Conjugate, which has been pre-coated in the test device). The mixture then migrates upward on the membrane chromatographically by capillary action to react with rK39 antigen on the membrane and generate a red line. The presence of this red line indicates a positive result while its absence indicates a negative result. Regardless of the presence of antibody to VL antigen, as the mixture continues to migrate across the membrane to the immobilized chicken anti-protein A region, a red line at the control line will always appear. The presence of this red line serves as a verification for sufficient sample volume and proper flow and is a control for the reagents.

Precaution during rK39 Test

The following points should be followed prior to testing and they are-

1. For professional in vitro diagnostic use only. Do not use it after the expiration date.
2. Handle all sera and kits used as if they contain infectious agents. Observe established precautions against microbiological hazards while performing all procedures and follow the standard procedures for proper disposal of sera and used kits.
3. Wear protective clothing, eye protection, and disposable gloves while performing the assay. Wash hands thoroughly when finished.
4. Avoid all contact between hands and eyes or mucous membranes during testing.
5. Do not eat, drink, or smoke in the area where the sera and kits are handled.
6. Chase Buffer contains a preservative; avoid all possible contact with skin and mucous membranes.

rK39 Test Requirements

1. Kit Contents: Kala-azar dipstick test strip is a membrane, pre-coated
   with a recombinant VL antigen on the test line region and chicken anti-protein A on the control line region. The Kit contains the following: Individually pouched test strips in a vial with desiccant in the cap and one vial of chase buffer solution.
2. Specimen: Serum/plasma or whole blood
3. Lancet ( for finger-prick optional)
4. Alcohol swab, dry cotton swab, and waste bin ( for finger prick)
5. Gloves
6. Timer
7. Centrifuge and test tube ( optional for serum/ plasma separation)

Test Procedure of rK39 Test

1. Wear gloves.
2. 1 or 2 drops of finger prick blood may be assayed for anti K39 IgG.
3. Remove the Kala-azar dipstick strip from the pouch or vial
4. Place one drop of blood on the absorbent pad on the stripped bottom.
5. Place the test strip into a test tube so that the end of the strip is facing downward.
6. Allow the mixture to migrate up to the strip by capillary action.
7. Add 2-3 drops of the Buffer solution provided with the test kit to the pad.
8. Read the results in 10 minutes. It is significant that the background is clear before reading the test, especially when samples have a low titer of the anti-Leishmanial antibody, and only a weak band appears in the test region (T). Results interpreted after 10 minutes can be misleading.

Result Interpretation of rK39 Test

Test positive: The test is positive when a control line and test line appear in the test area. A positive result indicates that the Kala-azar dipstick detected antibodies to L. donovani. A faint line is a positive result. The red color in the
test region will vary depending on the concentration of anti-Leishmanial
antibodies present. The test line for weakly positive sera samples may show results between a weak positive red line to a faintly red, almost white background. (Weakly positive samples are those with low affinity or low titer antibodies against the recombinant test antigen).

Test negative: The test is negative when only the control line appears. A
negative result indicates that the Kala-azar dipstick did not detect antibodies to L. donovani. An Invalid Result: If no lines appear at either the control or test line areas the

Test invalid: The test is also invalid if no control line appears, even though a
test line is seen. It is recommended to retest using a fresh dipstick and a fresh blood sample should be used in such a case.

Keynotes

1. There is a limited number of commercially available rapid diagnostic tests ( RDTs)  for Visceral leishmaniasis, such as the IT-LEISH (DiaMed AG, Switzerland – now Biorad, France), Kalazar Detect  (InBios International, USA), Onsite Leishmania Ab Rapid Test (CTK Biotech, USA) (Cunningham
   2012).
2. The sensitivity and specificity of tests vary manufacturer to manufacturer ( i.e. InBios to CTK Biotech) and even country to country.
3. Kalazar Detect™ Rapid Test(InBios International, Inc)’s sensitivity and specificity is 89.8% , 100% ( in Brazil) and 100 %, 93.12% ( India) respectively.
4. A study conducted by D. Singh et. al.’s ‘Novel Noninvasive Method for Diagnosis of Visceral Leishmaniasis by rK39 Testing of Sputum Samples’  result found very impressive compare to blood samples.
5. A study on ‘rK39 Antigen for the Diagnosis of Visceral Leishmaniasis by Using Human Saliva’ is also available.

Limitations of rK39 Test

1. This test will only indicate the presence of antibodies to the recombinant test antigen rK39 in patients with Visceral Leishmaniasis and should not be used as the sole criterion for the diagnosis of Leishmaniasis. This test alone must not be used for any clinical treatment decision. As with all diagnostic tests, all results must be considered with other clinical information available to the doctor.
2.  If the result is negative and clinical symptoms persist, additional follow-up testing using other clinical methods is recommended. A negative result does not preclude the possibility of Leishmaniasis.
3. A false-positive result may occur. Confirmatory testing (such as by culture) is advised especially in cases where no symptoms exist.
4. Do not use serum samples containing any glycerol or other viscous materials. This will decrease the sensitivity of the assay.
5. Persons with advanced HIV infection or other immunocompromising diseases frequently have low or undetectable anti-leishmanial antibodies.
6. This test may yield false-positive results with samples from patients having malaria.
7. The performance of this test has not been evaluated with L. infantum.
8. Certain Rheumatoid Factor (RF)-positive sera may produce false-positive results when Kalazar Detect™ is used.

Bibliography

1. Molecular Medical Parasitology. Editors: J. Joseph Marr, Timothy W. Nilsen and Richard W. Komuniecki, Publisher Academic Press, an imprint of Elsevier Science.
2. Merkell and Voge’s medical parasitology, 9th edition.
3. Parasitology: 12th edition by K. D. Chatterjee
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403768/
5. https://nvbdcp.gov.in/WriteReadData/l892s/Guidelines_on%20_use%20_of_rK39_rapid_diagnostic_kit.pdf
6. https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-017-2490-
7. https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-3156.2001.00680.x
8. https://www.who.int/medical_devices/diagnostics/selection_in-vitro/selection_in-vitro-meetings/FullSubmission_ID67_ID40.pdf?ua=1
9. https://jcm.asm.org/content/47/8/2684
10. https://ctkbiotech.com/product/leishmania-ab-rapid-test-ce/
11. http://inbios.com/wp-content/uploads/2016/06/900003-12-CE-Marked-Kalazar-Detect-Rapid-Test-Human-Insert.pdf
12. http://www.bioline.org.br/pdf?oc12200