Antibody/Antigen Detection Tests for the diagnosis of Kala-azar (Visceral Leishmaniasis)

Antibody detection tests

Antibody based tests must be used in combination with a standardized clinical case definition for Visceral Leishmaniasis diagnosis. 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-the direct agglutination test (DAT) and the rK39 based immunochromatographic test (ICT).

Direct Agglutination Test (DAT)

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 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):

Now 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; 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 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.

An rK39 based ELISA showed excellent sensitivity (93-100%) and specificity (97-98%) in many VL- endemic countries. The test is found in ICT or dipstick format, that are more suitable for field use.

Antigen-detection tests
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.