Elsevier

Acta Tropica

Volume 78, Issue 1, 15 January 2001, Pages 11-16
Acta Tropica

Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis

https://doi.org/10.1016/S0001-706X(00)00155-8Get rights and content

Abstract

This paper describes a new latex agglutination test (‘KATEX’) for the detection of leishmanial antigen in the urine of patients with visceral leishmaniasis. In preliminary laboratory trials, using urine collected from well-defined cases and controls from Brazil, Yemen and Nepal, the test had 100% specificity and a sensitivity between 68 and 100%. When used in a time-course experiment in cotton rats infected with Leishmania donovani, the test became positive 1 week after inoculation and antigen levels in urine declined rapidly after chemotherapy (the test was negative before the end of the course of treatment). Finally, in an integrated study performed in Sudan, KATEX was compared to microscopy and four different serological tests in a group of 73 patients having presented with clinical manifestations suggestive of visceral leishmaniasis. Compared to microscopy, KATEX performed better than any single serological test in predicting positivity and a particularly good result was obtained by combining KATEX and the direct agglutination test (DAT).

Introduction

Visceral leishmaniasis (VL), is a disease with an annual incidence of 500 000 cases worldwide and an estimated 200 million individuals at risk of contracting the infection (Ashford et al., 1992); the fact that 90% of clinical cases occur in the poorest communities in developing countries is an important consideration with regard to diagnosis and treatment. Clinical diagnosis relies on non-characteristic symptoms (cachexia, anaemia, chronic fever with hepato-splenomegaly) and is only reliable in advanced cases and in epidemic situations. Mortality of VL is high in the absence of treatment (using mostly injectable pentavalent antimony) which is lengthy (20–28 days in most regimes), very expensive and rather toxic. The finding of leishmanial amastigotes in stained smears from lymph nodes, bone marrow or splenic aspirates is generally accepted as the ‘gold standard’ for diagnosis, but the method is invasive, generally not feasible where is it most needed and it has poor sensitivity; while the in vitro cultivation of parasites from any of the above samples will improve sensitivity, this requires sophisticated laboratory facilities. Serological tests for the detection of antileishmanial antibodies are a well-developed diagnostic tool and a number of methods have been described, including indirect immunofluorescence (Badaro et al., 1983), ELISA (Hommel et al., 1978) and direct agglutination test (El-Harith et al., 1986). The use of recombinant leishmanial antigens (for example rK39; Houghton et al., 1998, Sundar et al., 1998) or synthetic peptide antigens (Fargeas et al., 1996) has recently been introduced for serology both in an ELISA and a dipstick format.

There are a number of problems with serological assays, including the possible cross-reaction with other pathogens including other Leishmania spp (responsible for cutaneous or mucocutaneous infections), Trypanosoma cruzi and mycobacteria and the fact that most serological tests cannot readily distinguish between current infection, subclinical infections or past infections. In an infection which remains asymptomatic in the majority of cases and where only an estimated 5–20% of infections ever become clinically patent (Hommel, 1999), this is a significant drawback to the use of serological tests in areas where infections are common, since seroconversion does not necessarily signify VL.

An antigen detection test would, in principle, provide better means for diagnosis since antigen levels are expected to broadly correlate with the parasite load. Antigen detection systems are also an ideal alternative to the antibody detection systems in immunocompromised patients and more particularly with the growing number of HIV co-infected cases, especially in advanced cases where the immune response is impaired (De-Gorgolas and Miles, 1994, Rosenthal et al., 1995). No satisfactory antigen detection test is currently commercially available and attempts to develop such tests have been unconvincing (Kohanteb et al., 1987, Senaldi et al., 1996).

This paper describes the development of an immunoassay to detect antigens in the urine of patients with visceral leishmaniasis and a preliminary field trial of its effectiveness.

Section snippets

Maintenance of Leishmania parasites

Leishmania donovani (MHOM/ET/67/HU3;LV9) were maintained in vivo in cotton rats (Sigmodon hispidus) by intraperitoneal sub-inoculation of amastigotes from infected cotton rats. Cotton rats are very susceptible to Leishmania infection and develop a heavy infection approximately 3 months after infection (Fulton and Joyner, 1948).

L. donovani promastigotes were maintained in vitro at 26°C in liquid HOMEM culture medium (Berens et al., 1976) containing 10% heat inactivated foetal calf serum (FCS).

Urine samples

Results

All urine samples from both endemic and European controls as well as VL patients from Brazil, Nepal and Yemen were tested using KATEX. Using this set of well-defined samples, the test had 100% specificity, since there were no cross-reaction with urine samples from endemic controls (zero positive out of a total of 57 samples tested). When the specificity was further evaluated by testing another 312 fresh urine samples from European negative controls from the Royal Liverpool Hospital, the initial

Discussion

In chronic infections, such as visceral leishmaniasis, the detection of antigens in patient serum is complicated by the presence of high levels of antibodies, circulating immune complexes (CIC), serum amyloid, rheumatoid factors and auto-antibodies, all of which may mask immunologically important antigenic determinants or competitively inhibit the binding of antibodies to free antigen. This may explain why no antigen detection assay for VL is routinely in use to-date, despite a number of

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