Research Article: Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard

Date Published: March 25, 2015

Publisher: Public Library of Science

Author(s): Sarah J. Wynwood, Mary-Anne A. Burns, Glenn C. Graham, Steven L. Weier, David B. McKay, Scott B. Craig, L. C. Pamela.

Abstract: A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays.

Partial Text: Leptospirosis is considered to be the most widespread zoonotic disease in the world [1] with clinical diagnosis proving challenging due to the non-specific nature of symptoms associated with the disease. There are some 300 leptospiral serovars belonging to a number of different serogroups. Currently there are 24 sero-groups of pathogenic leptospires based on their antigenic relatedness [2]. Leptospirosis was first reported in Australia in 1933 in the state of Queensland and has since been isolated Australia wide [3] with Queensland reporting the majority of these cases (57.6%) [4]. In 2011 the reported incidence of leptospirosis in Queensland was 3.4 cases per 100,000 people and overall in Australia the incidence was 0.84 cases per 100,000 people [5]. At present, 24 serovars of Leptospira spp are recognised in Australia and in recent years a dramatic increase in the incidence of leptospirosis cases in Australia (particularly Queensland) has been noted with environmental factors believed to be the main influence on this increase [6].

The aim of diagnostic serology is to determine reactive and non-reactive samples for a particular infectious agent. By definition, a validated assay consistently provides test results that identify samples as being reactive or non-reactive for a selected analyte, and, by inference, accurately predicts the disease status of patients with a predetermined degree of statistical certainty [21]. The aim of this study was to validate a microsphere immunoassay (MIA) using Luminex xMap technology for diagnostic leptospirosis serology screening. The validation process was performed using a comparative method—that is comparing the new assay with the current gold standard assay. Sixteen leptospiral antigens have been coupled to 16 individual magnetic bead-sets and validated as a panel for routine diagnostic leptospirosis serology. This assay gives a qualitative result—Reactive, Equivocal or Non-Reactive and has the ability to determine recent from past infection by differentiating between IgM and IgG antibodies—something that is more difficult to achieve with microscopic agglutination testing (MAT) as this test can only determine total antibody. The class of antibody detected by the MIA can be used to determine the stage of the infection which is valuable for clinicians as it can determine treatment regimens for patients or in the case of a past infection, can suggest that something other than leptospirosis is causing symptoms. Information regarding new infections is also vital from a public health perspective as it can provide information on what serovars of leptospirosis are currently circulating and indicate the areas where these infections are occurring.



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