Academic journal article Bulletin of the World Health Organization

Evaluation of the One-Point Microcapsule Aglutination Test for Diagnosis of Leptospirosis

Academic journal article Bulletin of the World Health Organization

Evaluation of the One-Point Microcapsule Aglutination Test for Diagnosis of Leptospirosis

Article excerpt

Introduction

Leptospirosis, a zoonosis with a worldwide distribution, is an acute febrile illness caused by microorganisms of the genus Leptospira. A diagnosis of human leptospirosis should be considered for all patients who present with an unknown disease with acute fever. The first leptospiraemic phase is characterized by acute systemic infection and by the presence of leptospires in the blood and cerebrospinal fluid under darkfield microscopy. This phase lasts 4-7 days; however, it is difficult to diagnose the disease at this stage. From the eighth day onwards (seldom earlier), serovar-specific antibodies may be demonstrated in blood serum by the microscopic agglutination test (MAT)[1], which is most widely employed as the standard reference test because of its high specificity and sensitivity. Currently, over 200 pathogenic leptospira serovars have been identified[2]. MAT requires multiple serovars of live leptospira, which involves a risk of infection and maintenance of a large number of stock cultures to provide antigens. These factors limit the test's usefulness for routine application in diagnostic laboratories.

We have developed microcapsules (MCs) of a synthetic polymer for use as an antigen carrier and reported a passive MC agglutination test (MCAT) for detecting antibodies to leptospires[3]. The high specificity and sensitivity of the MCAT to IgM antibodies make it suitable for the early diagnosis of leptospirosis. Furthermore, since the test is cross-reactive to antibodies from many members of the Leptospira genus, it may have an application as a genus-specific test[4]. A later development has been its use as a single-dilution (about 1:300) screening test for the early diagnosis of leptospirosis (5).

As a preliminary study to determine whether the MCAT could have an application outside Japan, we used it to demonstrate leptospiral antibodies in human serum samples from China, Republique of Korea, and Italy. The one-point MCAT gave encouraging results with the serum samples from China and Republic of Korea, but was less promising with the samples from Italy, possibly because of the presence of a greater number of serovars in Italy than in China and Republic of Korea[6, 7]. Other data obtained from Italy have been more promising, however, although the one-point MCAT failed to detect antibodies to serovar bratislava[8].

Subsequently, the test has been evaluated by six of the WHO Collaborating Centres for Reference and Research on Leptospirosis, and we report the results in this article.

Materials and methods

One-point MCAT

The MCAT [kit.sup.a] consists of two vials of lyophilized reagents (A and B) and one vial of a diluent (1% bovine serum albumin-PBS, pH 7.2). Reagents A and B are microcapsules sensitized separately with a mixture of sonicated antigens of Leptospira australis, L. autumnalis, and L. hebdomadis (reagent A); and L. canicola, L. icterohaemorrhagiae, and L. pyrogenes (reagent B). A test-tube rack with an oblique mirror and a number of disposable test-tubes are also provided with the kit.

The test procedure was as follows. After the reagents were reconstituted with the designated amounts of diluent, 0.3 ml of each reagent was transferred to corresponding tubes marked A or B. A sample of the test serum (about 1 [mu]1) was then added to both test-tubes and diluted about 300-fold. The tubes were mixed well and allowed to stand in the rack at room temperature for 3 hours. The reaction outcome was classified as 3+, 2+, +, [+ or -], or - according to the settling patterns. A reaction classified "+" was considered positive.

Leptospiral serovars used in the MAT

In the Russian Federation, strains responding to 13 serogroups were used in the MAT; the serum samples were positive to the following serovars: canicola, grippotyphosa, copenhageni, pomona, sejroe, and wolffi. In France 19 serovars were used: australis, autumnalis, castellonis, bataviae, canicola, cynopteri, grippotyphosa, hebdomadis, copenhageni, icterohaemorrhagiae, javanica, panama, pomona, pyrogenes, hardjo. …

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