A firm diagnosis of kala-azar/ visceral leishmaniasis (VL) requires demonstration of the parasite in splenic or bone marrow aspirate. But getting biopsy sample is an invasive test so different antigen/antibody detection methods are in use for the diagnosis of visceral leishmaniasis.
Antibody detection tests
Antibody based tests must be used in combination with a standardized clinical case definition for the diagnosis of visceral leishmaniasis. Serological tests based on indirect fluorescent antibody (IFA), Enzyme Linked Immunosorbent Assay (ELISA) or Western Blot have shown high diagnostic accuracy in most studies but are poorly adapted for field settings. Two serological tests have been specifically developed for field use and have been sufficiently validated are –direct agglutination test (DAT) and rK39 based immunochromatographic test (ICT).
Direct Agglutination Test (DAT)
Direct agglutination test (DAT) is another widely used test for serodiagnosis of kala-azar and is based on antigen-antibody reaction. Trypsin treated, stained and formalin preserved promastigotes are used as antigen which show agglutination with specific antibodies present in patients serum. The test is performed at room temperature though the antigens are stored under controlled temperature in freezer.
The usefulness of the above mentioned serological tests is limited by, their variable sensitivity or specificity, 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 grass roots in conjunction with the clinical diagnosis.
rK39 (Recombinant K39) Test:
rk39 test (popularly known as K39 test) is a rapid, non invasive test used for the diagnosis of visceral leishmaniasis (kala-azar). rK39 immunochromatographic tests, dipstick tests are easily available in endemic countries.
K39 is an epitope ( 39 amino acid repeats encoded by a kinesin-like gene ) apparently conserved on amastigotes of Leishmania species that cause visceral infection. By use of laboratory ELISA testing, circulating anti-K39, IgG is detectable in 95%- 100% of patients who have kala-azar, irrespective of geographic region.
Using K39 antigen-impregnated nitrocellulose strips developed for field conditions, fingerstick-obtained blood and serum samples demonstrated a sensitivity of 100% and a specificity of 97%. The strip testing proved simple to perform and yielded results within five minutes.
K39 strip test ( ICT or dipstick format) is ideal for rapid reliable field diagnosis of visceral leishmaniasis. An rK39 based ELISA showed excellent sensitivity (93-100%) and specificity (97-98%) in many VL- endemic countries.
A heat stable, low molecular weight carbohydrate antigen is detected by latex agglutination test in VL patients. It showed good specificity but only low to moderate (48-87%) sensitivity. Work to improve the format of this test is ongoing.