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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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Recently a new latex agglutination test (KAtex) based on antigen detection in urine has been introduced for the diagnosis of VL. Animal experiments and early results suggest that its a promising diagnostic tool, and its results correlate well with the splenic parasite status.7,8 Preliminary studies using urine of infected animals and patients have demonstrated the presence of leishmanial antigen in active VL, and its disappearance after successful treatment.7 Recently in two studies from Spain in 49 and 12 patients coinfected with HIV/VL, the sensitivity of KAtex was 85.7% and 100%, respectively,9,10 and the specificity in one of these studies was 96%.10 However, in another study from Nepal in large number of patients, the sensitivity was low (47.7%), although the specificity was high (98.7%).11 In this prospective study, KAtex has been evaluated for the diagnosis of patients with VL.
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MATERIALS AND METHODS |
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Collection and processing of urine. Patients and healthy subjects (controls) were asked to provide freshly voided urine in 20-mL test tubes, and 1 mL of urine was transferred to an Eppendorf (Hamburg, Germany) tube. The tubes were put into a tube holder and placed into a boiling water bath for five minutes, and then allowed to cool to ambient temperature before conducting the test.
Test kit. The test kit consisted of polystyrene (800-nm diameter) latex particles coated with polyclonal antibodies (against the leishmanial antigen),7 Leishmania culture supernatant diluted in saline as a positive control, and buffered saline as a negative control (both were preserved with sodium azide and ready to use). The kit also contains a reusable black glass slide with four equal circles (reaction zone) drawn on it for performing the agglutination (Kalon Biologic Ltd, Alder-shot, United Kingdom). After each use, the glass slide was rinsed with water and the surface was wiped with tissue paper. With one test kit, it is possible to test 100 samples including positive and negative controls.
Test procedure. All reagents were stored at 4°C and brought to ambient temperature. The latex particles were shaken immediately before use. Fifty microliters of the boiled urine was added to the reaction zone on the glass slide and one drop of latex was added. Both liquids were mixed into a completely homogenous mixture so that it covered the whole surface of the reaction zone. The glass slide was tilted with a rotating action in clockwise and anticlockwise directions alternately for two minutes, and the degree of agglutination was read. For every assay, a negative control was run in a reaction zone next to the test samples to distinguish between weak positive and negative results. The positive control was used periodically to monitor the performance of the test latex particles.
Interpretation of results. The degree of agglutination was recorded as follows: Positive +++ = the latex particles have agglutinated and much has collected around the edge of the reaction zone; positive ++ = the agglutinated particles can be clearly seen against a background of granular latex particles; positive + = agglutination can just be discerned when compared with the negative control. The degree of agglutination does not correlate with disease severity. When no agglutination was seen, the KAtex result was considered negative.
Urine samples from 382 parasitologically proven VL cases at diagnosis (baseline) were evaluated. All patients who opted for treatment at our center were provided a standard of care treatment with amphotericin B (Sarabhai Chemicals, Vadodara, India) in 1 mg/kg infusions on alternate days up to a total of 15 infusions. Splenic aspirates for post-treatment parasitologic evaluation were obtained one day after the last infusion of the drug. Of the 333 patients with a positive KAtex test result, 273 were treated at our center. The remainder either opted to receive treatment elsewhere or could not be accommodated. Thus, urine from these 273 patients who responded successfully to chemotherapy was available for testing immediately after the end of treatment. Freshly voided urine was also collected for testing from 185 healthy controls who lived in areas endemic for VL and were asymptomatic and had no abnormalities as obtained by a detailed clinical history and physical examination.
Diagnostic evaluation. Specificity, sensitivity, and predictive values of the assay system were calculated using the Bayesian approach.14 A kappa value was calculated to estimate the agreement of results between the gold standard for diagnosis and KAtex. The Fleiss Quadratic method was used for the calculation of confidence interval of proportions.
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RESULTS |
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. The response to antileishmanial therapy (amphotericin B) was prompt and post-treatment splenic aspirate smears of all patients showed no parasites. There was a reduction in spleen size, and a recovery in leukocyte, hemoglobin, and platelet levels (Table 1).
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DISCUSSION |
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The high specificity and positive predictive value of the KAtex test suggest that it may have an advantage over the DAT or rK39 strip test in which a greater proportion of controls from disease-endemic areas show a positive reaction. In this study, patients with other diseases such as tuberculosis or malaria were not included as controls. In future studies, it will be interesting to determine whether there is cross-reactivity between VL and other diseases.
This test is useful in difficult field conditions because it is simple, easy to perform, does not require any equipment, and is read visually. Although it has not been approved in any country, the test kit is commercially available from the manufacturer at a price of US $1.8 per test, which is slightly higher than rK39 strips (US $1.2–1.5). The disadvantages of the KAtex test include the requirement that the kit be stored at 4°C and its availability as a kit only for a minimum 100 tests. Conversely, rK39 strips and their buffer can be stored at room temperature and are available in single test kits. The need to boil the urine in field in the Katex test can also be difficult.
An added advantage of the KAtex test is that at the end of treatment it correlates well with the negative splenic parasite status in a high proportion of patients, and the results are positive in only a small number (3%) of patients. Its correlation with active VL makes it superior to other diagnostic test that use body fluids. Although DNA detection by the polymerase chain reaction is another very important tool in diagnosis and prognosis of VL,16 its high cost and requirement of sophisticated and expensive equipment and skilled personnel prevent its field applicability.
The absence of agglutination in 13% of the parasitologically positive patient urine samples could be due to a low amount of antigen. Improvements are needed to make this test more useful in disease-endemic regions. These include elimination of the need to boil the urine and refrigerate the reagents, and a more objective interpretation of the results. Nevertheless, the KAtex test appears to be an important advance in the non-invasive diagnosis of VL.
Received June 28, 2004. Accepted for publication February 18, 2005.
Acknowledgment: We are grateful to the nursing and technical staff of the Kala-Azar Medical Research Center (Muzaffarpur, India) for their participation and help in conducting the study.
Financial support: This work was supported by World Health Organization//World Bank/United Nations Development Program Special Programme for Research and Training in Tropical Diseases (ID 99106).
Disclosure: None of the authors have any conflicts of interest.
* Address correspondence to Shyam Sundar, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India. E-mail: shyam_vns{at}satyam.net.in 
Authors’ addresses: Shyam Sundar, Shrinkhla Agrawal, and Kalpana Pai, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India, Telephone: 91-542-236-7795, 91-94152-28390, and 91-542-231-2138, Fax: 91-542-236-7568, E-mails: shyam_vns{at}satyam.net.in, nehshri{at}yahoo.com, and kalpnapai{at}yahoo.com Michael Chance and Marcel Hommel, Liver-pool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom, Telephone: 44-151-705-3232 and 44-151-705-3169, Fax: 44-151-705-3371, E-mails: mchance{at}liv.ac.uk and mhommel{at}liverpool.ac.uk.
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