Diagnosis of Visceral Leishmaniasis by Enzyme-Linked Immunosorbent Assay Using Urine Samples
Shamsuzzaman, S. M.
Shamsuzzaman Choudhury, A. K. M.
Leafasia, J. L
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A diagnostic method has been developed to detect anti-Leishmania donovani immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-L. donovani IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using L. donovani promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated L. donovani promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of L. donovani crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-L. donovani IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-L. donovani IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.Visceral leishmaniasis (VL) is caused by protozoan parasites of the genus Leishmania and is transmitted by an insect vector, the phlebotomine sandfly. More than 47 countries are currently affected by leishmaniasis, with at least 200 million people at risk and approximately 100,000 new cases annually (2). Ninety percent of the cases occur in Bangladesh, India, Nepal, and Sudan. Bangladesh alone contributes about 15,000 new cases annually (10). Both the disease incidence and its severity—it is lethal if left untreated—are linked to poverty: malnutrition is associated with 8.7-times-higher risk for VL (7). Since the disease occurs mainly in areas where health services are poorly developed, the development of a simple, cheap, and reliable diagnostic method is necessary. Demonstration of the parasites in bone marrow aspirates or needle biopsy specimens of the spleen and lymph node or by vitro cultivation are the definitive methods of diagnosis (27). However, these methods are insufficiently sensitive, and the techniques are invasive, painful, and even hazardous (24). A number of serological tests have been developed and evaluated for the diagnosis of VL, including immunofluorescent-antibody tests (40, 43), enzyme-linked immunosorbent assay (ELISA) (12, 14, 15, 20, 22, 25, 33, 42, 46), dot ELISA (23, 31, 36), immunoblot analysis (28, 34, 35), and the direct agglutination test (DAT) (6, 17, 18, 19, 29, 30, 32, 41). Due to its simplicity and high sensitivity and specificity, the DAT has already been introduced as a routine serological test for diagnosis of VL in India and Bangladesh, and the parameters of the test have been established under local conditions (1, 8, 9, 10, 13, 41).In general, urine samples can be collected more easily than serum samples. To take advantage of this, several immunodiagnostic methods using urine have been established for some other diseases, like filariasis (21) and schistosomiasis (37). Kohanteb et al. (26) reported the detection of soluble antigen and antibody in the urine of VL patients by double-countercurrent immunoelectrophoresis, and de Colmenares et al. (11) detected antigenic compounds in urine by the Western blot technique. More recently, a latex agglutination test for the detection of Leishmania donovani antigen in urine was reported with good specificity but with sensitivity similar to that of microscopic diagnosis (3). In this paper, we report a sensitive and specific ELISA to detect anti-L. donovani immunoglobulin G (IgG) in urine using acetone-treated promastigote antigen.